Plasmodium berghei: II. Immunobiological properties of fractions of a soluble extract

A soluble extract of Plasmodium berghei was prepared from parasites freed from infected erythrocytes by saponin lysis. The extract was separated into 12 fractions by preparative disc electrophoresis, and the fractions were employed (1) to seek precipitins in hyperimmune rat serum, and (2) in the vaccination of rats. Species-specific antigens were identified in some of them.Two regions of the disc-electrophoretic column (Fractions 4–5 and 7–8) were regularly the most reactive in all systems tested. Thus, they reacted most frequently by precipitating with hyperimmune rat sera containing protective antibody, while other fractions were nonreactive or only rarely reactive. Antigens in these disc-electrophoretic regions were among those inducing precipitins in rats, though they were not the only ones to do so. These regions contained species-specific antigens, not shared with the noncross-protecting Plasmodium vinckei. Finally, fractions from these regions employed as vaccines were capable of inducing immunobiological effects: enhancement or protection in varying limited degrees.     

Plastein Reaction

α-Glucosidase (α-d-glucoside glucohydrolasc; EC: 3.2.1.20) has been extracted from bovine spleen and separated into two fractions (Fractions A and B) by gel filtration on Sephadex G-200. Fraction B which was retarded on Sephadcx columns contained only an acid α-glucosidase. This enzyme catalyzed hydrolysis of maltose and glycogen at comparable rates. Glycogen was hydrolyzed almost completely to glucose. The results of heat-treatment and of inhibition bv turanose demonstrated that Fraction A contained both acid if and neutral α-glucosidases. The neutral enzyme in Fraction A was further separated into four different components on preparative polyacrylamide gel electrophoresis. All of these neutral enzymes showed similar catalytic properties each other, and hydrolyzed maltose much more rapidly than glycogen. The acid enzyme in Fraction A was inactivated on the electrophoresis and was not further characterized.     

Protein composition of human submandibular secretions

The protein composition of human submandibular saliva obtained from a single donor has been investigated, and 21 proteins have been resolved. On Sephadex G-100, submandibular secretions (unstimulated) separated into four fractions, I, II, III, and IV. Each fraction was analyzed further by isoelectric focusing and disc gel electrophoresis. The major components detected in each fraction along with their isoelectric point (pI) are as follows: I, blood group specific substance (2.3), immunoglobulin A (5.0–6.0), and immunoglobulin G (4.5–6.5); II, albumin (4.9), two glycoproteins (5.0), and acid phosphatase (5.2); III, three phosphoproteins (4.3–4.4), isoamylase 1A (5.9), isoamylase 1B (6.4), unidentified protein (7.1), lysozyme (>10), and a basic protein (>10); and IV, isoamylase 2A (5.9) and isoamylase 2B (6.4). Isoamylase 1A and IB are glycoproteins. Stimulated submandibular secretions were also resolved into four protein fractions by gel filtration. Fraction III, compared with unstimulated secretions, showed the greatest percent increase in protein. Analysis of this fraction by disc gel electrophoresis demonstrated the presence of four protein bands which were not detected in the unstimulated secretion. One of these proteins is tentatively identified as a phosphoprotein and two as basic proteins (pI > 10). The protein composition of submandibular, parotid, and sublingual secretions is compared.     

The Partial Characterization of the Major Seed Storage Proteins in Arabidopsis thaliana L.

This study was aimed at the characterization of the major storage proteins in Arabidopsis thaliana. Two major protein fractions, i.e., the fraction Ⅰ and Ⅱ proteins, were isolated from the extract of mature seeds of this plant by molecular seive gel filtration chromatography. Various polyacrylarnide gel electrophoretic techniques were used to study the properties and polypeptide compositions of these two protein fractions. In was shown that during the SDS gel electrophoresis, fraction Ⅰ protein was separated into 6 major bands with the mol. was. of 34, 31, 29, 28 and 19-20 kD, respectively, whereas Fraction Ⅱ protein migrated as 3 low mol. wt. bands (10-12 kD) on the same gel. Non-denaturing native gel electrophoresis revealed that fraction Ⅰ was a neutral protein and Fraction Ⅱ was a positively charged basic protein with an isoelectric point (pI) higher than 8.8. Fraction I protein was further separated into at least 16 polypeptides in isoelectric focusing/SDS two-dimensional gel electrophoresis, i.e. each SDS band contained 3-4 polypeptides with the same mol. wt. but different pis. This suggested a more complex polypeptide composition of this protein. The properties of fraction Ⅰ and Ⅱ proteins were in good accordance with that of the 12s and 1.7s storage globulins in seeds of many other dicotyledonous plants, and therefore had been characterized as the two major seed storage proteins in this species. These two storage globulins were shown to be accumulated within a defined period during the late stage of seed development (12-14 DAF) and became predominant protein components in mature seeds. In the mean time, a few points in relation to the polypeptide composition and subunit molecular configuration of the 12s globulin were noted.     

Isolation and partial characterization of two glycoproteins from human liver metastases of sigmoid colon carcinoma

1. Perchloric acid-soluble glycoprotein fraction (PASF) extracted from human liver metastases (LM) of sigmoid colon carcinoma was chromatographed on a DEAE-cellulose column. The main fraction (DEAE-nonadsorbed fraction) passed through the column was then subjected to Sephacryl S-200 superfine gel filtration and separated into 12 fractions. 2. Among 12 fractions, only both Fractions 3 and 4 were demonstrated to be chemically and immunologically homogeneous glycoproteins, respectively, by a combination of chemical composition analysis, SDS-PAGE and EITB assay using antisera against the DEAE-nonadsorbed fractions of PASFs from human LMs, normal liver (NL) and normal sigmoid colon (NSC). Each of Fractions 3 and 4 reacted with anti-LM serum to give one immuno complex on a nitrocellulose sheet in EITB assay, but did not react with anti-NL and -NSC sera. 3. Apparent molecular weights of 80,900 and 62,100, respectively, were found for Fractions 3 and 4. Both the fractions, respectively, had abnormal sugar compositions. Fraction 3 contained sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine, but lacked glucose and mannose, and Fraction 4 contained sialic acid, fucose, galactose and N-acetylglucosamine, but lacked glucose, mannose and N-acetylgalactosamine, as sugar components.     

Partial separation of the pituitary proteins of Labeo umbratus, Smith (mudfish)

L. umbratus pituitary glands were partially separated by means of preparative polyacry-lamide electrophoresis and chromatography on DEAE-cellulose and Sephadex G-50. Eight fractions were obtaìned and it was found that fraction 1 contained potent adrenocorticotropic stimulating activity, with a molecular weight in the region of 6000. Fractions 3 and 4 contained potent gonadotropic and a lesser degree of exopthalmic producing activity, with molecular weights in the region of 14 000 to 20 000. Fraction 6 displayed pigeon-crop stimulating activity, with a molecular weight above 30 000. Fractions 7 and 8 displayed M.S.H. and vasopressinoxytocic activity respectively, with a molecular weight in the region of 5500–6500. Fractions 2 and 5 could not be identified. The fractions seemed to be glycoproteins and the results are discussed in relation to previous findings.     

Schistosoma mansoni: antigenic heterogeneity of excretions and secretions

The excretory-secretory antigens of adult Schistosoma mansoni were obtained by in vitro cultivation of worms in a chemically-defined medium. The protein output in this system was low, 0.2–0.4 μg of proteinworm/48 hr. The composition of the crude culture antigen (CA) was approximately 80% protein, 15% carbohydrate and 5% nucleic acid. Disc gel electrophoresis of CA revealed the presence of at least 15 protein components, many with carbohydrate moieties. Three major fractions were obtained by gel filtration on Sephadex G-200. Fraction I contained the bulk of the glycoprotein material. Immunoelectrophoresis of CA with hyperimmune rabbit serum indicated the presence of at least 6 antigens, most of which eluted in Fraction II. Serum from infected mice and monkeys, but not from rabbits and rats, reacted with CA and its fractions, especially Fraction II, on immunodiffusion analysis. Comparison of CA with other adult worm extracts by immunodiffusion techniques showed that most of the excretory-secretory antigens could be obtained by either freezing and thawing or by extraction with 3 M KCl. The P.K.-type activity of CA was considerably greater than that of a lipid-free adult worm antigen. Both Fractions I and II had the P.K.-type activity. An antigen capable of eliciting macrophage migration inhibition factor from infected rat lymphocytes was detected in CA, although the lymphocyte toxicity of CA was high at concentrations above 10 μg/ml.     

Studies on Barley and Malt Amylases

From the 2 m urea extract of ground barley two zymogen β-amylase (Z-β-A) fractions (Fractions A and B) and one active β-amylase (A-β-A) fraction (Fraction C) were isolated by Sephadex G-75 gel filtration and purified by DEAE-Sephadex A-50 column chromatography. The molecular weights of Fractions A, B and C were estimated to be approximately 280,000, 160,000 and 56,000, respectively.

Both the Z-β-A fractions which were ultracentrifugally and electrophoretically homogeneous were found to be accompanied by a small amount of saccharogenic activities. From the estimation of Km values for these saccharogenic activities and the behavior in their activation with 2-mercaptoethanol and papain, it seems reasonable to conclude that Fraction A is a heteropolymer type of Z-β-A composed of both A-β-A and barley reserve proteins; that Fraction B is a homopolymer type of Z-β-A composed of A-β-A alone; and that two different activation mechanisms, proteolytic activation and disulfide bond cleaving activation, are necessary for full activation of Z-β-A in barley.     

Effect of Individual Fractions Released from Myofibrils by CAF (Ca2+-activated Factor) on Z-Disk Reconstitution

To investigate the exact protein constituents of 2-disk on the basis of the success or failure of reconstitution of Z-disk, proteins released from myofibrils by CAF(Ca²⁺-activated factor) were fractionated, the Z-disk was reconstituted by incubating individual fractions with Z-disk- extracted fiber bundles and the proteins in each fraction were analyzed by polyacrylamide gel electrophoresis.Released materials from myofibrils by CAF were divided into three fractions, A, B and C, in the order of elution from a Sepharose 6B column. The materials in Fractions A and B have been bound in the Z-disk region, and the Z-disk extracted from myofibrils in a low ionic strength solution has been reconstituted. The Z-disk reconstituted by incubating the materials in Fraction A with Z-disk-extracted myofibrils seems to have a structure similar to the intact Z-disk. Fraction A consisted principally of proteins having subunit molecular weights near 100,000 and 34,000 daltons.     

Molecular Weight Determination of Gliadin Fractions in Gel Filtration by SDS-Polyacrylamide Gel Electrophoresis and Sedimentation Equilibrium

Gliadin was fractionated into three fractions; ω-gliadin, Fraction III (γ-gliadin) and Fraction IV (α- and β-gliadin). The determination of the molecular weights (MW) of the three fractions was performed by both SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and sedimentation equilibrium. In SDS–PAGE, ω-gliadin gave three bands (MW 50,000, 54,000 and 64,000), Fraction III two bands (MW 38,000 and 46,000) and Fraction IV two bands (MW 33,000 and 38,000), The sedimentation analysis showed that each fraction was fairly homogeneous relative to molecular weight. The molecular weights obtained by sedimentation were 28,000 for Fraction III and 27,000 for both Fraction IV and ω-gliadin. The disagreement in molecular weight between sedimentation and gel electrophoresis was discussed.     

A multicomponent protein of a fission yeast that promotes joint molecule formation from homologous DNAs.

We developed a quantitative assay ("homologous pairing gel assay") adequate for the purification of the activity promoting the formation of joint molecules, an intermediate of homologous recombination ("homologous pairing"). With this assay, one can measure the extent of homologous pairing between a single-stranded DNA and a strand of 3H-labeled double-stranded DNA by crude enzyme preparations. Since the total activity did not significantly change during the sporulation process, we tried to purify the activity from a whole cell extract of mitotic cells of a fission yeast, (Schizosaccharomyces pombe). Through quantitative assaying of a single fraction or of mixed fractions, we obtained three fractions, all of which were required for the maximum level of the ATP-independent homologous pairing: Fractions 65, 100, and 30. In Fractions 100 and 30, polypeptides of approximately 100 and approximately 30 kDa (the 100- and 30-kDa polypeptides), respectively, were the sole detectable components. Fraction 65 contained a polypeptide of approximately 65 kDa (the 65-kDa polypeptide) as the major component and also small amounts of the 30- and 100-kDa polypeptides. Fraction 65 by itself promoted homologous pairing, but the reaction was saturated at a level of approximately 20% of the maximum level achieved with the recA protein. Even when added in excess, Fraction 30 or 100 alone did not promote detectable homologous pairing. A mixture of Fractions 65 and 100 at a rather strict optimum ratio only promoted homologous pairing, the level being 50-70% that with the recA protein, suggesting a stoichiometric complex of these polypeptides as the active form. Fraction 30 alone did not enhance the reaction with Fraction 65, but stimulated homologous pairing promoted by the optimum mixture of Fractions 65 and 100 to the maximum level achieved with the recA protein. Therefore, the homologous pairing-promoting protein from the fission yeast is likely to be a multicomponent protein.     

ISOLATION OF COATED VESICLES, PLAIN SYNAPTIC VESICLES, AND FLOCCULENT MATERIAL FROM A CRUDE SYNAPTOSOME FRACTION OF GUINEA PIG WHOLE BRAIN

Two vesicular fractions and one nonvesicular fraction were prepared from crude synaptosomes by differential centrifugation and salting out with ammonium sulfate. Fraction 1 contained a mixture of coated vesicles, material thought to be derived from breakdown of the coats (shell fragments), and plain synaptic vesicles. Fraction 2 contained a mixture of plain synaptic vesicles and flocculent material. Fraction 3 contained flocculent material only. Fractions 1 and 3 were partially purified by passage through a Sephadex column. Fraction 3 contained no shell fragments but contained finer flocculent material which, it is suggested, is composed of unit particles either occurring singly or linked together into chainlike or amorphous aggregates. Each unit particle appears to have four subunits and is here referred to as a tetrasome. Tetrasomes sometimes appear to be attached to the surfaces of the plain synaptic vesicles. Also, it is possible that aggregates of tetrasomes form part of the structure of the presynaptic dense projections.     

Isolation and characterization of Chromatium vinosum membranes

A fractionation of Chromatium vinosum into an outer layer (cell wall) and three intracellular membrane fractions by isopycnic sucrose density centrifugation of a total membrane fraction obtained by lysis of lysozyme-EDTA spheroplasts is decribed. The three intracellular fractions (I, II, and III) have apparent densities of 1.11, 1.14, and 1.16, respectively, and contain the bulk of the photosynthetic pigments. Fraction II is enriched in bacteriochlorophyll and contains about 49% of the total membrane protein and 60% of the membrane bacteriochlorophyll. The outer membrane fraction (IV, cell wall) has a density of 1.23 and contains 5% of the membrane protein and 0.8% of the bacteriochlorophyll. Fraction I is enriched in lipids and phosphorus and has only a trace of diaminopimelate (DAP). Fractions II and III both contain a significant content of DAP. Fraction IV has no DAP, but has a fatty acid composition similar to that of the envelope fraction. Electrophoresis of the fractions on sodium dodecylsulfate-containing gels yielded from 8–13 bands of protein. Fractions I, II, and III contained the same series of unique proteins, while fraction IV contained another group of unique proteins. In fraction IV the bulk of the proteins traveled in one band with a molecular weight of 41,500. Examination of the fractions and whole spheroplasts in the electron microscope showed that fractions I, II and III were composed of large membrane structures in the form of membrane reticulum with bud-like appendages, and elongated flattened tubes. Fraction IV was composed of large ovoid structures which were seen to lie on the outer surface of the whole spheroplasts. These results suggest that the normal in vivo state of the intracellular membranes is that of an interconnected series of tubules and vesicles extending throughout the cell cytoplasm.     

On the nature of the activating enzyme of the inactive form of delta-aminolevulinate synthetase in Rhodopseudomonas spheroides.

The activating enzyme of the inactive form of Fraction I of delta-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas (R.) spheroides was purified about 1,000-fold from an extract of R. spheroides cells grown anaerobically in the light. The purification of the activating enzyme was achieved by fractionating the 100,000 X g supernatant fraction of the crude extract with ammonium sulfate and acetone, followed by Sephadex G-200 chromatography, pyridoxamine phosphate-Sepharose 4B chromatography, and preparative gel electrophoresis. The final preparation of the activating enzyme still contained a minor contaminant (less than 20%) as judged by disc gel electrophoresis. The activating enzyme exhibited cystathionase [EC 4.4.1.1] activity throughout the purification. These two enzyme activities were not separated at all during any step of the purification. An apparently homogeneous preparation of cystathionase [EC 4.4.1.8] purified from rat liver also exhibited activating activity in the presence of L-cystine. It was concluded that the activating enzyme is a cystathionase.     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of two antigenic glycoproteins from the pollen of Prosopis juliflora

Two antigenically active glycoprotein fractions were isolated from crude extract of the pollen of Prosopis juliflora using DEAE-cellulose ion exchange chromatography. The glycoproteins gave single band on polyacrylamide gel electrophoresis. The molecular weight of these two glycoprotein was 20,000 and 10,000 as determined by gel filtration on Sephadex G-75. With the help of crossed immunoelectrophoresis and gel diffusion crude extract exhibited twelve and three precipitating antigens suggesting its heterogeneous nature; and the purified glycoprotein fractions however formed single precipitin band on gel diffusion test and immunoelectrophoresis. As tested by ELISA the polyclonal antisera raised in rabbit showed strong binding affinity with glycoprotein of MW 20,000. These result indicates that the two glycoprotein fractions are not antigenically identical.     

alpha-Amylase Isozymes in Gibberellic Acid-treated Barley Half-seeds

The presence of multiple forms of α-amylase in gibberellic acid-treated embryoless barley half-seeds was demonstrated by separation on diethylaminoethyl-Sephadex and isoelectric focusing polyacrylamide gel disc electrophoresis. Two major α-amylase fractions (A and B), each consisting of two to three isozyme components, were purified. α-Amylase fractions A and B were distinguishable in their reaction patterns. The optimal pH of fraction A α-amylase was found to reside in the acidic side (pH 5.0), as was determined by analyzing the reducing sugars formed as well as the paper chromatographic detection of reaction products. At neutral pH, 6.9, fraction A exhibited weak amylolytic activity in forming maltose. The α-amylase activity in fraction A was markedly stimulated by heat treatment (70 C/15 minutes). Fraction B, constituting a major part of amylases in the endosperm extract, was also found to be composed of α-amylase, as evidenced by the loss of enzyme activity upon allowing fractions A and B to stand at pH 3.3 for a prolonged period. The possible physiological function of the two different types of α-amylase in the carbohydrate breakdown of barley seeds is discussed.     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

THE SURFACE FEATURES OF DROSOPHILA EMBRYONIC CELL LINES

Schneider's cell lines 1, 2 and 3 derived from embryonic stages of Drosophila melanogaster were examined by scanning electron microscopy, histochemical staining procedures, and SDS polyacrylamide disc gel electrophoresis. Although the three lines showed some similarities when compared by each of these methods, differences between the lines were observed as well. The surface features of the cells in each line showed morphologic as well as age dependent distinctions when examined in the scanning electron microscope. The intracellular distribution and amount of periodic acid-Schiff positive (PAS+), α-amylase sensitive material was also distinct for each of the lines. Approximately one-third of line 1 cells contained PAS+ hyaluronidase-sensitive material localized within a cytoplasmic vesicle whereas less than 1% of the cells of lines 2 and 3 contained such material. SDS polyacrylamide disc gel electrophoresis revealed PAS+ material in a soluble fraction of line 2 cells and in the 100,000 μ g pellet of line 3 cells, but no PAS+ bands in similar fractions of line 1 cells were detected.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.