Evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical

Bovine erythrocyte green heme binding protein and bovine erythrocyte flavin reductase have been isolated in highly purified forms and subjected to amino acid analysis and N-terminal amino acid sequence analysis. The two proteins possess similar amino acid compositions and identical N-terminal amino acid sequences. Moreover, the two proteins are immunochemically cross-reactive and are indistinguishable when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by double diffusion technique. This study provides evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.     

Mass spectrometric sequencing of endotoxin proteins of Bacillus thuringiensis ssp. konkukian extracted from polyacrylamide gels

The amino acid sequences of the crystal proteins of Bacillus thuringiensis ssp. konkukian strain HL-47 are unknown. We used 1-D denaturing polyacrylamide electrophoresis, nano-ESI-Q-TOF-MS, and protein database searching to analyze these proteins. On SDS-PAGE gels, a preparation of purified crystal proteins exhibited 110, 102, 76, 55, 37, and 30 kDa protein bands. Immunoblotting of the gel with antiserum raised to this preparation revealed that four crystal proteins, of 110, 102, 55, and 37 kDa, reacted with the specific antiserum. The 102-kDa major protein reacted strongly. The other crystal proteins showed weak immunoreactivity. The 102 and 55 kDa proteins were analyzed by ESI-MS. The internal amino acid sequence of the 102-kDa major protein has similarity to the sequences of the surface layer protein of B. thuringiensis ssp. finitimus and B. anthracis. However, the internal amino acid sequences of the 55 kDa protein did not show any homology to proteins in the databases. Proteomic analysis of these proteins leads to the conclusion that the sequence data provided the protein databases of the crystal proteins of the konkukian ssp.     

Purification and characterization of the major sialoglycoproteins of the rat erythrocyte membrane

The major sialoglycoproteins of the rat erythrocyte membrane were purified by hot phenol partitioning followed by cation-exchange chromatography on SP-Sephadex. Further purification was obtained by extraction with n-butanol and anion-exchange chromatography on DEAE-cellulose. The resulting sialoglycoprotein fraction was free of lipids and nonsialylated glycoproteins and gave rise to four major periodic acid-Schiff staining bands when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fastest migrating protein on these gels with an apparent molecular weight of 19,000 was purified to homogeneity by gel filtration. The amino acid and sugar compositions of these materials are reported. The protein moiety is rich in serine, threonine, and hydrophobic amino acids and the carbohydrate moiety is high in sialic acid and N-acetylgalactosamine. Most of the carbohydrate is linked O-glycosidically to serine and threonine residues, as shown by susceptibility to base-catalyzed β-elimination and concomitant reduction of serine and threonine to alanine and α-aminobutyric acid and of N-acetylgalactosamine to N-acetylgalactosaminitol in the presence of reducing agents. The significance of these data in light of the known role of the rat erythrocyte membrane sialoglycoproteins in erythropoiesis is discussed. The properties of the rat erythrocyte membrane sialoglycoproteins are compared to those of other species.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Studies on Stigma Pellicle Glycoproteins of Raphanus sativus L.

Stigma surface diffusates of Raphanus sativus were subjected to polyacrylamide gel electrophoresis. There appeared protein bands, one of which gave positive PAS reaction, indicating it was glycoprotein. SDS polyacrylamide gel electrophoresis showed that the stigma diffusates contained many protein bands. By comparing their mobilities with those of standard proteins of known molecular weights, the molecular weights of some of the major fractions were estimated to be 15000, 30000—46000 and 70000 daltons. After Schiff reagent staining two main glycoprotein fractions appeared on the SDS gel electrophoretic pattern. Their molecular weights were estimated to be lower than 15000 and higher than 100000 daltons. By using acrylamide gel isoelectric focusing method, it was found that the stigma surface diffusates contained an acidic glycoprotein with pH of about 3.7. The amino acid composition of the purified stigma glycoprotein was determined with amino acid analyzer. Glycine, glutamic acid, serine, aspartic acid were some of the predominant amino acids. The diffusates were analysed by gasliquid chromatography for sugars. Results showed that the carbohydrate fraction of the glycoprotein consisted of arabinose 17.3%; galactose 19.1%, xylose 8.1%, mannose 5.4%, glucose 23.7%, rhamnose and/or fucose 26.4%. In the stigma surface diffusates of Raphanus sativus, the content of protein was estimated to be 16% and that carbohydrate was 11%.     

Partial characterization of the 30 kD Ig-binding protein from Pseudomonas maltophilia.

We have previously demonstrated that Pseudomonas maltophilia (ATCC 13637) possess a 30 kDa cell wall protein which binds various subclasses of IgG's and IgA by their Fc region. The protein was solubilized by papain and purified by affinity chromatography on cyanogen bromide activated sepharose beads conjugated with human IgG. The eluent was electrophoresed on a 12% polyacrylamide gel under denaturing conditions, and the immunoactive bands identified by Western blot analysis, a second gel was stained with Coomassie blue. The affinity purified eluent was electrophoresed on a one-dimensional 15% polyacrylamide gel and stained with Coomassie blue. The protein band of interest was cut. The protein band was then digested in situ with Staphylococcus aureus V-8 protease. The peptide bands were separated by electrophoresis on a second one dimensional 15% polyacrylamide gel and then electroblotted into a polyvinylidine difluoride membrane. The bands were visualized by staining with Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Minimal amino acid composition was determined in a similar fashion. We have thus obtained partial N-terminal amino acid sequence data from the above method.     

Amino acid compositions of simian virus 40 structural proteins

The structural proteins of purified SV40 particles were isolated by preparative polyacrylamide gel electrophoresis and the amino acid composition of each protein was obtained. The amino acid composition of VP1 (the major coat protein) was significantly different to that of VP3 (the capsid protein most closely associated with SV40 DNA). The amino acid compositions of VP4, VP5 and VP6 indicated that these proteins were not exclusively histones.     

Methyl acceptors for protein methylase II from human-erythrocyte membrane.

Membrane proteins from human erythrocytes were methylated with purified protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC.2.1.1.24). The methylated proteins were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Monomeric and dimeric glycophorin A (NaIO4/Schiff-2 and NaIO4/Schiff-1 positive bands) and 'band 4.5' were identified as two major classes of methyl-acceptor polypeptides for protein methylase II. In rabbit erythrocyte membrane where glycophorin A is absent, 'band 4.5' was the only major methyl-acceptor protein component. Extracted and purified glycophorin A from human erythrocytes was also found to be an excellent substrate for protein methylase II with a Km of 35.7 microM. The role of erythrocyte membrane protein methylation is discussed with regard to membrane function.     

A comparative study of the polypeptides of three iridescent viruses by N-terminal analysis,amino acid analysis,and surface labeling

Polyacrylamide gel analysis of the structural proteins of three types of iridescent viruses (2, 6, and 9) demonstrated that the purified virions had one major and more than 20 minor polypeptides. Surface labeling procedures performed on pure intact virions, using ¹²⁵I in the presence of lactoperoxidase and chloramine T (at low iodine concentrations), demonstrated that the major and two or three minor polypeptides were located on the outside. The major structural polypeptide was isolated from each virus type by preparative polyacrylamide gel electrophoresis. Amino acid analysis indicated that this protein was very similar in the three iridescent viruses. The three polypeptides had an identical N terminal (proline). While the major polypeptide of each virus has a slightly different molecular weight as determined by polyacrylamide gel electrophoresis, the similarities in iodine labeling, N terminals, and amino acids suggests a common function for this protein.     

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

Brassica napus plastid and mitochondrial chaperonin-60 proteins contain multiple distinct polypeptides.

Plastid chaperonin-60 protein was purified to apparent homogeneity from Brassica napus using a novel protocol. The purified protein, which migrated as a single species by nondenaturing polyacrylamide gel electrophoresis, contained four polypeptides: three variants of p60cpn60 alpha and p60cpn60 beta. Partial amino acid sequence determination demonstrated that each variant of p60cpn60 alpha is a distinct translation product. During this study, additional chaperonin-60 proteins were purified. These proteins, which were free from contaminating plastid chaperonin-60, were separated into at least two high molecular weight species that were resolved only by nondenaturing polyacrylamide gel electrophoresis. These proteins contained three 60-kD polypeptides. Two of these polypeptides were recognized by existing antisera, whereas the third was not. Partial amino acid sequence data revealed that each of these, including the immunologically distinct polypeptide, is a chaperonin-60 subunit of putative mitochondrial origin. The behavior of chaperonin-60 proteins during blue A Dyematrex chromatography suggests that this matrix may be generally useful for the identification of chaperonin-60 proteins.     

Purification and characterization of placental protein 5

This report describes the purification of placental protein 5, PP5, from the human placenta by two affinity chromatography steps, the first with Heparin-Sepharose and the second with Sepharose-linked monoclonal anti-PP5 antibody. The final purification is achieved by reversed-phase high performance liquid chromatography. In SDS-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, PP5 purified in this study migrates as one major band at 36 kD. The previously purified PP5 is more heterogeneous: under nonreducing conditions it migrates at 30 kD and, after reduction, it gives three bands at 16.8 kD, 18.3 kD, and 19.0 kD. In Western blot analysis, both purified proteins react with polyclonal and monoclonal anti-PP5 antibodies. Three N-terminal amino acid sequences are obtained for the previously purified PP5, whereas the N-terminal of PP5 purified in this study is blocked. These results suggest that PP5 previously purified in the absence of protease inhibitors, does not represent the native form of PP5. Computer comparison of the obtained amino acid sequences revealed no significant homology to known protein sequences.     

Purification and properties of a predominantly female-specific protein from the hemolymph of the larva of the tobacco hornworm, Manduca sexta

A major serum protein was isolated from the hemolymph of larvae of the female tobacco hornworm, Manduca sexta, just prior to metamorphosis. After 3 or 4 days, this predominantly female-specific protein is rapidly cleared from the hemolymph and taken up and stored by the fat body. This larval serum protein was purified by density gradient ultracentrifugation, gel permeation, and ion-exchange chromatography. The purified protein exhibits a single band on native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chemical cross-linking with dimethylsuberimidate indicates a hexameric subunit arrangement for the native protein. The amino acid composition, relatively rich in methionine but poor in cysteine, was used to calculate a v = 0.75 cm3/g. Analytical ultracentrifugation experiments yielded S020,w = 16.9 S and D020,w = 3.23 X 10(-7) cm2/s. From these values Mr = 510,000, f/f0 = 1.22, and Stokes radius = 66.3 A were calculated. Immunoblotting experiments with anti-larval serum protein serum indicate a cross-reactivity with storage protein-1 of Bombyx mori. The amino acid composition and immunological data suggest that larval serum protein may be an example of a class of insect storage proteins distinct from the arylphorins, which are characterized by high content of aromatic amino acids.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

Characterization of soluble protein extracts from keratinized tissues: identification of ubiquitin universally distributed in hair, nail, and stratum corneum

Partial protein extracts were prepared from hair, nail, and stratum corneum in the absence of urea and interfacial surfactant. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoreses of these extracts showed low-molecular weight protein-rich patterns apparently different from those of whole protein extracts, which mainly consist of keratin bands. Several protein bands characterized each keratinized tissue or its derived species. In addition, we identified a major band of approximately 7 kDa as ubiquitin, a ubiquitously distributed protein that mediates non-lysosomal protein degradation, through direct amino acid sequence analysis of the electro-blotted protein band. The partial extraction is useful for investigation of soluble proteins retained in the keratinized tissues.     

Poly(ADP-ribose) polymerase from Ehrlich ascites-tumour cells. Amino acid composition, N-terminal analysis and chemical cleavage of the purified protein.

Poly(ADP-ribose) polymerase was purified from Ehrlich ascites-tumour cells by two novel methods. Analysis for amino acid composition revealed a high percentage of acidic amino acids or their amides, and of basic amino acids. N-Terminal analysis with dansyl chloride revealed no terminal amino acid, indicating a blocked N-terminal amino group. Analysis by gel electrophoresis of protein treated with 3-bromo-3-methyl-2-[(2-nitrophenylthio)-3H-indole, under conditions where selective cleavage of the polypeptide chain at tryptophan residues is obtained, showed six major peptide bands.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.