Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting.

Polyacrylamide gel electrophoresis of lysates of purified Rickettsia tsutsugamushi revealed as many as 30 polypeptide bands, including major bands corresponding to molecular sizes of 70, 60, 54 to 56, and 46 to 47 kilodaltons. Compared with the polypeptide composition of the rickettsiae of Gilliam, Karp, and Kato strains and a newly isolated Shimokoshi strain, the major polypeptide in the Kato strain (54-56K) and in the Karp strain (46-47K) migrated a little faster and slower, respectively, than the corresponding polypeptides in the other strains. The largest major polypeptide (54-56K) was digestible by the treatment of intact rickettsiae with trypsin and variable in content in separate preparations, suggesting that the polypeptide exists on the rickettsial surface and is easily degraded during the handling of these microorganisms. Several surface polypeptides of rickettsiae, including the 54-56K and 46-47K polypeptides, were detected by radioiodination of intact rickettsiae followed by polyacrylamide gel electrophoresis of the lysate; however, the 70K and 60K polypeptides were not labeled. Immunoblotting experiments with hyperimmune sera prepared in guinea pigs against each strain demonstrated that the 70K, 54-56K, and 46-47K polypeptides showed antigenic activities. The 54-56K polypeptide appeared to be strain specific, whereas the 70K and 46-47K polypeptides cross-reacted with the heterologous antisera.     

Rubella virion polypeptides: Characterization by polyacrylamide gel electrophoresis,isoelectric focusing and peptide mapping

Summary Four polypeptides with molecular weights of 55K, 47K, 45K, and 33K have been resolved by polyacrylamide gel electrophoresis of immune precipitated rubella virus. The 47K and 45K components have similar peptide maps but different isoelectric points so that the same polypeptide may exist in more than one charged form. The 55K and 45K components have similar isoelectric points but different peptide maps showing that similarity of isoelectric point is not evidence of identity.With 4 Figures     

Characterization of a coronavirus: I. Structural proteins: Effects of preparative conditions on the migration of protein in polyacrylamide gels

Coronavirus A59 possesses four size classes of structural proteins which have apparent molecular weights measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 23,000 (GP23), 50,000 (VP50), 90,000 (GP90), and 180,000 (GP180). VP50 is the only structural protein which is completely unaffected by protease treatment of intact virions. This species is the most highly labeled by polar amino acids such as glutamic acid and arginine and it is probably associated with the viral nucleocapsid. GP90, GP180, and GP23 are membrane-associated proteins. However, after protease treatment of virions, only 20% of the GP23 molecule is digested, whereas all of the GP90 and GP180 are removed. GP90 and GP180 appear to comprise most of the prominent layer of characteristic projections on the external surface of the viral envelope. The major portion of GP23 is presumed to lie within the lipid envelope, protected from protease digestion. GP23 and the protease resistant portion, p¹18, exhibit anomalous behavior on SDS-PAGE. After heating to 100° in SDS the electrophoretic mobility of these polypeptides is altered and several new forms of lower mobility are produced. β-Mercaptoethanol and dithiothreitol exaggerate the effects of heating.     

Comparison of synaptic plasma membrane and synaptic vesicle polypeptides by two-dimensional polyacrylamide gel electrophoresis.

Extracts of chick brain synaptic plasma membranes, synaptic vesicles, and mixtures of membranes and vesicles were examined by electrophoresis on two-dimensional polyacrylamide gels by a modification of the O'Farrell technique. Synaptic plasma membranes had twenty-one major polypeptides; synaptic vesicles had seventeen. Thirteen major polypeptides were common to both fractions. The similarities between the synaptic vesicle and synaptic plasma membrane patterns are unlikely to be due to contamination of one fraction by the other or to contamination of both fractions by microsomes, synaptoplasm or mitochondria. Our findings are consistent with mixing of membrane proteins occurring during exocytosis but it remains to be shown that these synaptic subfractions are not contaminated by a type of membrane for which markers are not yet available.     

Human influenza A virus: comparative analysis of the structural polypeptides by two-dimensional polyacrylamide gel electrophoresis.

The biochemical properties of the major virion polypeptides (HA₁, HA₂, M, NP, NA) of 19 influenza A virus strains have been compared by two-dimensional polyacrylamide gel electrophoresis using nonequilibrium pH gradient electrophoresis in the first dimension. The highly variable surface antigen of the virus, the hemagglutinin (HA), exhibited multiple polypeptide subspecies varying extensively in charge. Comparison of the HA of the different influenza A strains demonstrated that most strains exhibit a unique hemagglutinin with distinguishable electrophoresic properties. Differences in charge and/or molecular weight of at least one of the two HA subunits (HA₁ or HA₂) were found for strains within the same subtype and for serologically indistinguishable strains such as A/USSR/90/77 and A/FW/l/50. Differences in the matrix (M) and neuraminidase (NA) proteins also were observed between strains. The results of this study indicate that the comparative examination of the two-dimensional polypeptide patterns of a particular virus isolate may be useful for the purposes of strain identification, determination of strain purity, homogeneity, and determination of gene origin following experimental or natural recombination events.     

Identification of orthopoxviruses by polyacrylamide gel electrophoresis of intracellular polypeptides. I. Four major groupings.

Orthopoxviruses were compared by means of their late intracellular polypeptides (ICPs). HeLa cells were labeled with [³⁵S]methionine at 16 hr after infection and the ICPs were analysed by SDS-polyacrylamide gel electrophoresis and autoradiography. Time course and pulse-chase studies confirmed the significance of the differences observed. ICPs in three regions of the gels (MW 177,000, 96–103,000, and 15,500) permitted an initial grouping of the 24 viruses examined into four main groups (cowpox, monkeypox, vaccinia, and variola-related viruses). Only cowpox-like viruses had a prominent ICP of MW 177,000, and only monkeypox-like viruses had a heavily labeled ICP of MW 15,500. Viruses in these two groups appeared to have only a single ICP in the 96–103,000 region (MW, 98,000). By contrast, vaccinia-like viruses and variola-like viruses had two ICPs in this region but were distinguished because the ICPs of vaccinia were of higher molecular weight and were apparently reversed with repect to those of variola-like viruses. This was supported by the disappearance on chase of the lower MW vaccinia ICP and the higher MW variola ICP. The significance of these results and their use in the identification of orthopoxvirus isolates, particularly whitepox viruses, has been discussed.     

Demonstration and characterization of polyoma-tumor-associated antigenic components using preparative and analytical polyacrylamide gel electrophoresis

Glycoproteins from surface of polyoma tumor cells extracted with 3M KCl were separated in preparative electrophoresis and elution profile showed eight fractions, which were further separated by polyacrylamide gel electrophoresis and SDS-PAGE. Twenty polypeptides were detected, their molecular weights were in range 10000-155000 but most of them had Mr 17000-68000 and isoelectric points ranged between 3.9 and 5.5. Results of immunodiffusion after absorption of immune sera suggested that tumor associated antigens were present in fraction II in which four glycopeptides of molecular weights 14,000, 67,000, 36,000, 18,000 were found. These polypeptides had isoelectric points 5.3, 5.9, 6.4 and 6.6 respectively. Antigens of H-2 system were detected in fraction IV, which contained three glycopeptides of molecular weights 18000, 37000 and 48000 and isoelectric points 6.0, 5.8, and 6.7 respectively.     

Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice.

Pathogenicity of Rickettsia tsutsugamushi for laboratory mice is known to be influenced by at least three factors: (i) route of inoculation, (ii) antigenic strain, and (iii) natural resistance of the host. By using Karp, Gilliam, and Kato strains of R. tsutsugamushi, we examined the effect of these three pathogenicity factors on the kinetics of infection and the development of immunity in BALB/cDub and C3H/HeDub mice. The appearance of rickettsemia in the pathogenic infections generally preceded infections of reduced pathogenicity by 1 to 2 days in both magnitude and time of onset. Mice infected by the subcutaneous route with normally pathogenic rickettsiae, i.e., Gilliam-infected C3H/HeDub mice and Karp-infected BALB/cDub mice, consistently maintained a detectable rickettsemia over a 1-year period. Rickettsiae were recovered from the spleens of 95% (19 of 20) of these mice 52 weeks postinfection. In contrast, mice with infections of reduced pathogenicity, i.e., BALB/cDub mice infected by intraperitoneal and subcutaneous inoculation with Gilliam, did not have detectable rickettsemia from week 20 through week 52 postinfection except for a single mouse on week 44 postinfection. Rickettsiae were detected in the spleens of only 40% (8 of 20) of these mice after 1 year. In both Gilliam-infected mouse strains, protection against heterologous challenge with Karp or Kato rickettsial strains was incomplete up to 7 days postimmunization. Infections of reduced pathogenicity did not result from an enhanced systemic immune response by the host. The onset of the humoral response was not different for the pathogenic and reduced-pathogenicity infections. Pathogenicity differences seemed to result from the more rapid growth of the rickettsiae in the pathogenic infections.     

Characterization of sodium dodecyl sulfate-stable Bacteroides gingivalis proteases by polyacrylamide gel electrophoresis.

Profiles of the proteolytic activities found in Bacteroides gingivalis culture supernatants, outer membranes, vesicles, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing covalently bound bovine serum albumin. A total of eight distinct bands of proteolytic activity could be detected. Four of these were found in the culture supernatant (P1, P2, P3, and P4). The outer membranes, vesicles, and the cell extract each contained seven major proteolytic bands (P1, P3, P4, P5, P6, P7, and P8). No activity was found in the membrane-free extract, suggesting that the proteases were associated with the cell envelope. With the exception of P7 and P8, all the proteolytic bands were dependent on reducing agents for activity. The eight proteolytic bands were distributed in an identical manner in all four strains of B. gingivalis studied. The effects of protease inhibitors, pH, and heat were determined. Sulfhydryl group reagents and N-alpha-p-tosyl-L-lysine chloromethyl ketone reduced proteolytic activity. The optimum pH was found to be between 7 and 8. A 30-min preincubation at 50 degrees C inactivated the P6, P7, and P8 proteolytic bands. All proteolytic activity was lost after the samples were heated at 75 degrees C for 30 min.     

Unusual migration profiles of Japanese encephalitis virus structural polypeptides in SDS-polyacrylamide gel electrophoresis

Summary The migration profiles of JEV polypeptides in SDS-PAGE are influenced by electrophoretic conditions. The estimated molecular weights of N and M became smaller when lower gel concentration and longer electrophoretic run were adopted.With 3 Figures     

One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria widely used as a fluorescent probe. In this study, phycoerythrin of the red macroalga Palmaria palmata was extracted by grinding the algal sample in liquid nitrogen, homogenisation in phosphate buffer and centrifugation. Phycoerythrin was then purified from this crude extract using preparative polyacrylamide gel electrophoresis (PAGE) with a continuous elution system and detected by its pink colour and fluorescence. The pigment presented a typical spectrum of R-phycoerythrin, with three absorbance maxima at 499, 545 and 565 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A565/A280, a criterion for purity, was 3.2. A single protein of relative molecular mass 240,000 was detected on native-PAGE with silver staining. Sodium dodecyl sulphate-PAGE demonstrated the presence of two major subunits with Mr 20,000 and 21,000, respectively, and a very minor subunit of Mr 30,000. These observations are consistent with the (alphabeta)6gamma subunit composition characteristic of R-phycoerythrin. Phycoerythrin of Palmaria palmata was determined to be present in larger amounts in autumn and showed a good stability up to 60 degrees C and between pH 3.5 and 9.5. In conclusion, phycoerythrin of Palmaria palmata was purified in a single-step using preparative PAGE. Obtaining pure R-phycoerythrin of Palmaria palmata will allow one to evaluate its fluorescence properties for future applications in biochemical techniques.     

Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum.

Pseudomonas aeruginosa exotoxin has been purified to a specific activity of 12,000 to 16,000 mouse median lethal doses/mg of protein. Total recovery was about 25%, and the degree of purification was approximately 3,000-fold. Preparative polyacrylamide gel electrophoresis greatly facilitated purification. As judged by analytical disc gel electrophoresis, the purified toxin contained one major band of protein and only a negligible amount of contamination. Antiserum prepared against the purified toxin neutralized the lethal activity of crude toxin preprations and reacted by double immunodiffusion with a single component of concentrated broth cultures of P. aeruginosa isolates obtained from a clinical source.     

Molecular weight analysis of the polypeptides of cholera phage PL 163/10 by polyacrylamide gel electrophoresis.

Cholera phage PL 163/10, belonging to Mukerjee's Group I, was purified by alternate cycles of low and high speed centrifugation. Gel electrophoresis revealed the presence of four polypeptide chains of respective molecular weights of 10,370+/-515 (A), 30,000+/-1,303 (B), 40,000+/-1,049(C) and 64,000+/-2,433 (D) daltons. Electrophoresis of the sample alkylated with iodoacetic acid resolved the presence of only one polypeptide chain of an average molecular weight of 10,310+/-565 daltons. The polypeptides B, C and D could be interpreted as trimer, tetramer and hexamer respectively of the polypeptide A, which was the basic structural protein of this phage.     

Genomic study of Rickettsia akari by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of SmaI-, EagI-, and BssHII-digested DNA was used to perform restriction fragment length polymorphism analysis of Rickettsia akari strains isolated from humans, rodents, and mites in the United States and Ukraine. Although some differences in biological and serological characteristics were present between strains, the genomic studies demonstrated a high degree of intraspecies homogeneity of R. akari isolates. Our results confirm the value of pulsed-field gel electrophoresis-restriction fragment length polymorphism analysis for the identification of species of rickettsiae.