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The role of Β-glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by the phosphoglucomutase for Β-glucose 1-phosphate (Β-phosphoglucomutase) has been examined in purified extracts of Euglena gracilis var. bacillaris. 1. The incubation of Β-glucose 1-[32P]phosphate with Β-phosphoglucomutase in presence of high concentrations (0.1 mM) of a commercial preparation of α-D(+)-glucose 1,6-bisphosphate (prepared synthetically) yielded a labelled compound running electrophoretically and chromatographically as the sugar bisphosphate. Specificity studies with Β-phosphoglucomutase and muscle phosphoglucomutase (α-phosphoglucomutase) strongly suggest that the compound formed is Β-glucose 1,6-[32P]bisphosphate. The results would also indicate that the commercial preparation of α-D(+)-glucose 1,6-bisphosphate contains Β-glucose 1,6-bisphosphate as contaminant. 2. When synthetic Β-glucose 1-phosphate preparations were purified by paper chromatography and then incubated with Β-phosphoglucomutase, no glucose 6-phosphate could be detected. On the other hand the reaction readily took place when challenged with the chromatographic fraction that runs as sugar bisphosphate. Upon analysis with etectrophoresis, chromatography and weak acid hydrolysis, and experiments with Β-phosphoglucomutase and α-phosphoglucomutase it was concluded that this compound is Β-glucose 1,6-bisphosphate. 3. It was established that the natural α-D(+)-glucose 1,6-bisphosphate not only fails to sustain the reaction catalyzed by Β-phosphoglucomutase but rather inhibits the reaction when added to the whole Β-phosphoglucomutase system, it was also demonstrated that Β-glucose 1,6-bisphosphate acts as inhibitor of the α-phosphoglucomutase system. 4. These results show for the first time that Β-glucose 1,6-bisphosphate is an active and necessary participant in the reaction catalyzed by Β-phosphoglucomutase. The role played by Β-glucose 1,6-bisphosphate in this reaction would be essentially the same as that of the α-anomer in the reaction catalyzed by α-phosphoglucomutase. [ABSTRACT FROM AUTHOR]

Succinate dehydrogenase flavocoenzyme (‘SD-flavin’), previously shown to be an 8α-substituted riboflavin derivative containing a tertiary nitrogen homoconjugated to the flavin nucleus, was subjected to further hydrolysis and to reduction under acid conditions. Both conditions resulted in the liberation of 1 mole of histidine per mole of flavin. This proves histidine to be the covalent link between flavin and peptide backbone in succinate dehydrogenase and imidazole to be the tertiary nitrogen function homoconjugated to the flavin. 8α-Histidyl-riboflavin has been synthesized starting from riboflavin chemically and shown to be completely identical with the natural product in optical, ESR and NMR spectra, pH-fluorescence curve and behavior on thin-layer and paper chromatography, as well as paper electrophoresis. [This equation cannot be represented into ASCII Text] 3. Both the natural compound isolated by acid hydrolysis of flavin peptide and the synthetic one contain two isomers, which may be separated by high voltage electrophoresis. The isomers appear to be the N(1)- and N(3)-imidazole substituted compounds. Digestion of the flavin peptide with aminopeptidase M yields only one isomer but on treatment with 6-N HCl this is gradually converted to a mixture of the two isomers. The absolute assignment of the natural isomer is suggested as 8α-[N(3)-histidyl]-riboflavin on the basis of imidazole quaternization with CH3I, reductive cleavage of the flavin-imidazole bond and identification of the methyl-histidine liberated as 1-methyl-histidine. [ABSTRACT FROM AUTHOR]

The article presents information on various forthcoming papers to be published in the March 15, 1984 issue of the "European Journal of Biochemistry." Some of them are "Studies on the Structure and Expression of Escherichia Coli pyrC, pyrD and pyrF Using the Cloned Genes," "Rat-Liver Lysosomal Sialidase. Solubilization, Substrate Specificity and Comparison With the Cytosolic Sialidase," "Diamine-Induced Dissociation of the First Component of Human Complement," "Evidence for the Glycoprotein Nature of Retina Glycogen," and "Mapping of Surface Glycoproteins of Trypanosoma Cruzi by Two-Dimensional Electrophoresis. A Correlation With the Cell Invasion Capacity."

A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann). Purification was achieved by gel filtration on Sephadex G-25 and G-15, and preparative paper electrophoresis at four pH values (1.8, 3.6, 6.0 and 10.0). The recovery of hormone activity was 85% and the specific activity was 4.0 × 104 limes that of the starting material. The hormone is a thermostable peptide of approximately 1200 molecular weight and composed of neutral amino acids. No N-terminal group could be found. From its electrophoretic behavior it is concluded that the C-terminal group is also blocked. [ABSTRACT FROM AUTHOR]

The nitrogenous materials released by the blue-green phycobiont (Nostoc sp.) of five lichens have been examined by means of high voltage paper electrophoresis. They were found to be a mixture of substances with similar electrophoretic characteristics to the materials released by the same algae when grown in pure culture, and were largely peptide in nature. A more detailed study was made of the components released into their growth media by the three strains of Nostoc isolated from Peltigera aphthosa, P. canina and P. polydactyla. From about twelve compounds demonstrable by electrophoresis, the six most abundant were examined for their amino acid composition. The only finding strikingly different from existing data was a generally low level of serine. Molecular weight estimates by means of gel filtration indicated that most of the substances had molecular weights of approximately 1100, with substances with molecular weights of about 300 forming a smaller important group, although materials with a wide range of molecular weights were evidently present and low molecular weight material could also be important. [ABSTRACT FROM AUTHOR]

Low molecular weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudontoytas aeruginosa were isolated from the fraction containing the partially degraded polysaceharide, by successive chromatography on columns of Sephadex G-75 and G-10, followed as necessary) by preparative high voltage paper electrophoresis The major components identified were 2-keto-3-deoxyoctonic acid, basic amino acids (free and bound), inorganic orthophosphate, ethanolamine phosphate and ethanolamine pyrophosphate. Ethanolamine pyrophosphate has not previously been found in acid hydrolysates of lipopolysaceharides. Ethanolamine phosphate and some of the orthophosphate were apparently produced by breakdown of ethanolamine pyrophosphate [ABSTRACT FROM AUTHOR]

Peptide B, the N-terminal fragment of thioredoxin obtained by cyanogens bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues peptide B. The N-terminal sequence of peptide B was determined as: Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe. [ABSTRACT FROM AUTHOR]

The methylation of HeLa cell ribosomal proteins has been studied by paper electrophoresis separation of the peptides produced after trypsin digestion of individual ribosomal proteins. Methylation in vivo was detected by 3H labeling of peptides from cells that had been incubated with a mixture of [methyl-3H]methionine plus [35S]methionine, the incorporation of the 3H-labeled amino acid methionine being corrected by the 35S uptake. Each methylated peptide thus represented at least one methylation site (one or more methylated amino acids and their adjacent amino acid sequence). Eleven ribosomal proteins were found to be methylated. As many as five methylation sites were present in one ribosomal protein. In several ribosomal proteins some sites were methylated when ribosome synthesis was suppressed with low levels of actinomycin D, while other sites were not. Therefore, some of the sites in a given protein were apparently methylated in mature ribosomes, while other sites were methylated during ribosome processing. The methyl-group-specific labeling of some sites, but not others, was suppressed by cycloheximide, thus suggesting that the methylation of some sites was dependent on protein synthesis. A cell-free system from cultured HeLa cells is described, which reproduces the methylation pattern of ribosomal proteins in vivo in two ways: methylation was observed both in vivo and in vitro (a) in the same ribosomal proteins; and (b) in the same methylation sites of a given ribosomal protein. In addition, our data in vitro suggest that the methylation of some human ribosomal protein sites may involve a methyl donor other than S-adenosylmethionine. [ABSTRACT FROM AUTHOR]

Abnormally high amounts of low molecular weight mannose-rich carbohydrate material were found in the urine of an Angus calf with mannosidosis. At least five oligosaccharide fractions were detected by paper chromatography. The most abundant compound was purified by gel chromatography, zone electrophoresis, and two consecutive preparative paper chromatographic steps. The yield was 10 mg/liter of urine. From structural studies including nuclear magnetic resonance spectroscopy, optical rotation, sugar analysis, methylation analysis, and partial enzymatic degradation the following structure was deduced: α-D-Manp- (1 → 6)-β-D-Manp-(1 → 4)-β-D-GlcNAcp-(1 → 4)-β-D-GlcNAcp-(1 → 4)-D-GlcNAc. This oligosaccharide is distinct from all the oligosaccharides previously described which are excreted by patients with mannosidosis. [ABSTRACT FROM AUTHOR]

Mild acid hydrolysis of the glycolipid derived @om a Salmonella Rd2 mutant led to the liberation of three major split products which, after separation by electrophoresis, were identified as free 2-keto-3-deoxyoctonate (KDO), KDO-7-phosphorylethanolamine, and L-glycero-α-D-mannoheptosyl-l,5-KDO. [ABSTRACT FROM AUTHOR]

Activity of mammalian pyruvate dehydrogenase complexes from all sources so far tested is regulated by phosphorylation involving three sites. To facilitate understanding of the precise biological roles of the individual phosphorylation sites a method has now been developed, using pig heart [32P]phosphorylated complexes, which enables unambigous measurement of their occupancies. Established methods of tryptic digestion give two peptides that contain the three phosphorylation sites: TA contains sites 1 and 2; TB contains site 3. Thus, while occupancy of site 3 may be determined unequivocally by tryptic digestion, occupancies of sites 1 and 2 cannot. The present paper shows that peptide TA may be specifically and quantitatively cleaved by formic acid at an Asp-Pro bond located between the two phosphorylation sites. Equal amounts of two new peptides each containing a different phosphorylation site (site 1 or site 2) are produced. The 32P-labelled peptides may be completely separated and quantified by high-voltage paper electrophoresis at pH 2. A combination of tryptic digestion (determination of 32P in site 3) and-formic acid cleavage of peptide TA (determination of 32P in sites 1 and 2) thus enables unequivocal assignment of occupancies of individual phosphorylation sites to >95 % accuracy. This method has been used to show that during phosphorylation and inactivation of pig heart complexes (inactivated to between 1.5% and 90%) >98% of the observed inactivation was primarily attributable to phosphorylation of site 1; the contribution of site 2 was <2 % if at all. Relative initial rates of phosphorylation, site 1:site 2:site 3 were approximately 90:3:1. [ABSTRACT FROM AUTHOR]

The polysaccharide moiety of the lipopolysaccharide (polysaccharide I) from Escherichia coli F492 (08:K27-:H-) and the polysaccharide (polysaccharide II) from the rfa mutant F612 (derived from F492) were isolated by extraction with 450% aqueous phenol at 65 °C. Polysaccharide I was obtained in 1–2% yield (based on dry bacteria). It contained mannose (83.5%), glucose (5.7%), galactose (3.4%), heptose (4.6%) and 2-keto-3-deoxy-mannosulonic acid (0.8%). Polysaccharide II was obtained in 0.8–1% yield (based on dry bacteria). It contained 99.2% mannose. With the method of Yphantis it was found that the molecular weights of polysaccharides I and II were 12400 and 10400, respectively. The difference accounted for the core oligosaccharide which was present in polysaccharide I and absent in polysaccharide II. The specific optical rotation was 43 °C for polysaccharide I and 42 °C for polysaccharide II. Both polysaccharides were permethylated. Subsequent hydrolysis, reduction and acetylation, followed by gas-liquid chromatography and mass spectrometry indicated that in both polysaccharides two-thirds of the mannose units were substituted at C-2 and one-third at C-3. These results were confirmed by periodate oxidation. From polysaccharide II nine oligosaccharides were obtained after partial acid hydrolysis by chromatography on Biogel P2, paper chromatography and paper electrophoresis of the borate complexes. The oligosaccharides were reduced with sodium borodeuterate (which labelled the reducing mannose units) and methylated. After hydrolysis, reduction and acetylation the products were analyzed by gas chromatography and mass spectrometry. It is concluded that the poIysaccharides I and II contain about 20 repeating units of α-mannosyl-1,2-α-mannosyl-1,2-α-mannose which are joined through &alph;-1,3 linkages. [ABSTRACT FROM AUTHOR]

An inhibitor from Pinus radiata was separated using paper chromatography from an acidic ether fraction of a stem tissue extract. Using electrophoresis the inhibitory activity was resolved into two parts. One part remained at the origin. The second part behaved similarly to ABA, in electrophoresis and was investigated further. The inhibitor has similar properties to ABA in electrophoresis; TLC using a number of solvent systems; UV fluorescence spectra: UV absorption; ORD spectra and gas chromatography. Final proof of the identity of the natural inhibitor as ABA was obtained by gas chromatography-mass spectrometry. [ABSTRACT FROM AUTHOR]

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From the lipopolysaccharide of the gram-negative bacterium Rhodopseudomonas capsulata (Athiorhodaceae) a lipophilic sugar was isolated by chromatographic procedures and characterized by mass spectrometry as belonging to the group of 3-O-methyl-6-deoxy-hexoses. Demethylation showed that the parental sugar had the same properties in borate electrophoresis as rhamnose. Comparison with an authentic sample of 3-O-methyl-rhamnose in gas liquid chromatography, paper chromatography and borate electrophoresis showed that the sugar under examination is 3-O-methylrhamnose (acofriose). Investigation of its optical rotation indicates that the sugar has the L-configuration. L-Acofriose (3-O-methyl-L-rhamnose) has not hitherto been found in lipopolysaccharides of gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Describes the circumstances guiding the Sephadex group of products for gel filtration with reference to the importance of electrophoresis. Description of dextran-based gels; Information on Jerker Porath's experience from zone electrophoresis; Details on Porath's research of electrophoresis.

Reports on the isolation and characterization of the O-glycoside oligosaccharides from the major cell surface glycoprotein of Zajdela hepatoma ascites cells. Liberation of the oligosaccharides as alditols by alkaline borohydride treatment, gel filtration on Bio-Gel and paper electrophoresis; Yield of four oligosaccharide-alditols fractions; Structure characterization by nuclear magnetic resonance spectroscopy.

An inhibitor for S-adcnosyl-L-methionine (AdoMet)-dependent metbyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two- dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyitransferases examined. [ABSTRACT FROM AUTHOR]

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9–204, 56–66, 71–77, 121–193, 126–177, 134–149, 145–158 and 159–164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158. [ABSTRACT FROM AUTHOR]

From Escherichia coli 0124 two lipopolysaccharide preparations were obtained with phenol/water extraction and cetavlon precipitation. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and chemical analysis showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. In passive haemagglutination and gel precipitation the two lipopolysaccharide preparations from E. coli 0124 and the corresponding preparations from Shigella dysenteriae type 3 reacted alike. The O-specific polysaccharide moiety was characterized with proton magnetic resonance spectroscopy, optical rotation and paper electrophoresis. The constituents were determined by gas chromatography and ion-exchange chromatography. The polysaccharide contained glucose (Glc), gatactose (Gal), galactosamine (GalN) and 4-O-(1′-carboxyethyl)-D-glucopyranose (glucolactilic acid, GIcLA) in the molar ratios of 1 : 2 : 1 : 1. Glucolactilic acid, which has a structure similar to muramic acid, was first found in Sh. dysenteriae. The polysaccharide from E. coli 0124 and oligosaccharides obtained from it by partial acid hydrolysis were subjected to methylation analysis using the method of combined gas chromatography—mass spectrometry. The results indicated that the pentasaccharide repeating unit of the polysaccharide is[This symbol cannot be presented in ASCII format] In the polysaccharide the repeating units are joined through galactofuranosidic linkages. This structure is identical with that of the somatic polysaccharide of Sh. dysenterae type 3. [ABSTRACT FROM AUTHOR]

We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gets, exceeds 18 × 106, i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 × 106, i.e. twice the molecular weight of one mini-circle. The &Deltm of native and renatured duplexes is about 1°C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, get electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the ‘rosette’ structures and the long edge loops characteristic of intact kinetoptast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together: Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the mini-circles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence, Possible mechanisms that could account for the micro-heterogeneity in sequence of mini-circles are discussed. [ABSTRACT FROM AUTHOR]

Several large oligonucleotides obtained by partial digestion of tRNAIIArg from brewers' yeast with ribonnclease T1 have been isolated and their sequences have been established. These results and those described in the preceding paper permitted the derivation of the complete primary structure of this tRNA. [ABSTRACT FROM AUTHOR]

The positions of the disulfide bridges in toxin Il of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high-voltage electrophoresis. Determination of the amino acid compositions of the cystinecontaining peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half-cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins. [ABSTRACT FROM AUTHOR]

Glycoproteins were isolated from the ovary of the starfish Asterias rubens (L.). After delipidation, sugar analysis revealed the presence of mannose, glucose and N-acetylglucosamine in a molar ratio of 9.0:1.3:2.3. Subsequently, hydrazinolysis, re-N-acetylation, reduction and high-voltage paper electrophoresis were carried out, resulting in a mixture of neutral oligosaccharide alditols which was fractionated on Bio-Gel P-4. The alditols, investigated by 500-MHz ¹H-NMR spectroscopy, turned out to be of the oligomannose type or of the glucooligomannose type containing 9 mannose and 1-3 glucose residues. The most abundant compounds were established to be: Manα1 → 2Manα1 → 6 Manα1 →6 6 Manα1 → 2Manα1 → 3 Manβ1→ 4GlcNAcβ1 → 4GlcNAc-ol Manα1 → 2Manα1 → 2Manα1 → 3 and Manα1 → 2Manα1 → 6Man&alpha :1→ 6 Man&alpha:1 &rarr:2M anα1→3 Manβ1→ β1 → 4GlcNAc·ol Glcα1 → 3Manα1 → 2Manα1 → 2Manα1 → 3. [ABSTRACT FROM AUTHOR]

This paper is the first one of a series devoted to the primary structure of the major protein of cow's milk, αs1-casein, a single-chain phosphoprotein, formerly reposed to have arginine as an NH2-termimal, and Leu-TrpOH as COOH-terminal residues. ryptic digestion of maleyl αs1-casein (genetic variant B), in theory bruited to arginyl bonds, yields eight peptidic fragments, Tm1 to Tm8, which were fractionated by column chromatography and, in some cases, more thoroughly purified by preparative paper electrophoresis and/or chromatography. The ammo acid composition and the phosphate content of these fragments were determined. The fragments Tm3 and Tm8 arise from a partial non specific cleavage of Tm5. Thus, six fragments only—2, 4, 5, 6 and 7—arise from the specific cleavage of the αs1-casein chain at arginyl bonds, a result in accordance with the reported number of six arginyl residues in the chain, one being NH2-terminal. The fragment Tm7, with two arginyl residues, and Tm2 devoid of arginine appear to rcpresent respectively the NH2- and COOH-terminal parts of the αs1-casein chain. These peptidic fragments—except Tm4 which is devoid of lysine—were further digested by trypsin at lysyl bonds, after eliminating the blocking maleyl groups. The resulting peptides were purified by the same methods as mentioned above, and the composition of these tryptic peptides was established. As a control, the tryptic peptides of αs1-casein were simultaneously purified by the same methods from a hydrolysate of the protein which had not been treated with maleic anhydride. In summary, this first step of our work gives: (a) the composition of ail the bovine αs1-casein tryptic peptides; (b) partial information on the location of these peptides in the protein chain: (c) confirmation that the molecular weight of the bovine αs1-casein monomer is approximately 23600. [ABSTRACT FROM AUTHOR]

This paper reports a method of performing electrophoretic deposition (EPD) on non-conducting substrates overcoming the requirement of a conducting substrate through the use of porous substrates. The conductivity of the substrate is therefore no longer a limiting factor in the application of EPD. This method is applicable to the fabrication of thick or thin layers of ceramic or metal for various applications. As an example, thin and dense yttria-stabilized zirconia (YSZ) layers have been deposited on a non-conducting NiO–YSZ substrate by EPD from a non-aqueous suspension. A solid oxide fuel cell constructed on these sintered bilayers exhibited power densities of 384 and 611 mW/cm2 at 750° and 850°C, respectively. [ABSTRACT FROM AUTHOR]

Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+-dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate–polyacrylamide gel electrophoresis as a band of∼53 000 MW in the reduced state and∼138 000 MW in the unreduced state. Porcine SP-D was sensitive to collagenase digestion and N-deglycosylation, which reduced the molecular mass to∼24 000 MW and∼48 000 MW respectively, in the reduced state. N-deglycosylation of the collagen-resistant fragment, reduced the molecular mass to∼21 000 MW showing the presence of an N-glycosylation site located in the CRD. Porcine SP-D bound to solid-phase mannan in a dose and Ca2+-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP-D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkühn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland. [ABSTRACT FROM AUTHOR]

The present paper describes how epithelial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains α1(IV) and α2(IV). Metabolic labeling of these cells with [14C]proline for different rime intervals and subsequent analysis by SDS/polycrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. the procollagens were bacterial-collagenase-sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procolagen poly(A)-rich RNA was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell-free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of α,α'-bipyridyl, type IV procollagen appeared as one major comigrating with a 145 kDa polypeptide on SDS-gel electrophoresis. [ABSTRACT FROM AUTHOR]

1. In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P. M. and Holms, W. H. (1975) J. Gen. Micribiol. 87, 37-51]. 2. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. 3. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. 4. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of onedimensional peptide mapping. 5. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were directs of identical subunits. 6. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule subunit and the partially active form contained an intermediate amount. 7. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). 8. The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed. [ABSTRACT FROM AUTHOR]

The histone variants and high-mobility-group (HMG) proteins of a transcribing fraction of chromatin, described in the preceeding paper of this journal, have been analysed qualitatively and quantitatively by a combination of one-dimensional and two-dimensional gel electrophoresis. The stoichiometry of the four core histones (all variants included) in this fraction is equimolar and is not detectably different from that in the nontranscribing fraction or in total chromatin. The molar ratio of histone Hi to the core histones is markedly lower, by approximately 72%, than that in the nontranscribing fraction. A minor histone variant identified as Ml (an H2A variant) is detected only in the transcribing fraction, while variant 113.1 is found only in the non- transcribing fraction. Proteins A24, HMG1 and HMG2 are essentially absent from the transcribing fraction; HMG14 is found uniquely in this fraction, while HMG17 occurs at a relatively lower level. [ABSTRACT FROM AUTHOR]

Surface glycoproteins on plasma membranes from Dictyostelium discoideum were labeled by sodium metaperiodate oxidation and sodium boro[³H]hydride reduction. The amount of incorporation of tritium from NaB³H4 reached a plateau after 10 min at a periodate concentration of 20 mM. The density analysis carried out by sucrose density-gradient centrifugation showed that the plasma membranes were selectively labeled by this technique. About 84% of the radioactivity incorporated was released by hydrolysis with 0.25 M H2SO4 for 3 h at 100 °C. The released materials were eluted at a bed volume after chromatography using a Sephadex G-50 column. From the paper chromatographic analysis of the eluate, four radioactive spots were detected. Two of them were glycerol and glyceraldehyde and the other two spots seemed to be oligosaccharides. Using the above method, the plasma membranes from aggregation-phase cells were labeled four times more than those from growth-phase cells. The labeled plasma membrane fraction during the aggregation phase was separated into at least 39 distinct glycoprotein bands on a one-dimensional dodecylsulfate/polyacrylamide gel. Six of the 39 bands increased markedly in density and two new bands appeared. In contrast, four bands decreased in density in the aggregation phase. Using this method, the surface glycoproteins can be analyzed directly by gel electrophoresis of the lysate of labeled whole cells without preparing the plasma membranes. Changes during development of glycoproteins on outer cell surfaces were also confirmed by O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis. Utilizing this technique, glycoproteins on plasma membranes of D. discoideum were separated into 63 individual spots; 45 of these 63 spots changed during the early developmental course of D. discoideum. This is in contrast to the slight change in the soluble and membrane proteins during this phase. This fact also suggests that the glycoproteins have important roles in the process of aggregation. [ABSTRACT FROM AUTHOR]

A thermosensitive sporulation mutant (ts-15) of Bacillus subtilis has been isolated. This mutant when grown at the restrictive temperature (42 °C) is unable to sporulate, shows no intracellular protease activity and no protein turnover. These three traits were recovered in two revertants (ts-I 5R1 and ts-15R2) and were also transmitted together by transformation into the wild type. Immunological studies have shown that when ts-15 is grown at 42 °C it synthesizes a ‘cryptic’ protein with apparently the same antigenic properties as the wild type or as ts-15 mutant grown at the permissive temperature (30°C). The intracellular proteases from the wild type and from ts-15 grown at 30°C and 42°C were completely purified and their properties were studied with respect to their molecular weights, substrate specificity, inhibition pattern, heat inactivation and antigenicity. The molecular weight of the enzyme from the wild type or ts-15 grown at 30°C was 64000–65000 in the absence of sodium dodecylsulfate and 31000–32000 in the presence of sodium dodecylsulfate. It was assumed therefore that the active enzyme is formed from two similar subunits. However, the intracellular protease from ts-15 grown at 42°C showed the same molecular weight of 32000–34000 in the presence or in the absence of sodium dodecyisulfate. On the basis of this experiment and others described in the paper we concluded that the mutation in ts-15 is most likely a point mutation in a structural gene of an intracellular protease and results in an inability to assemble the two subunits into an active form. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

A comparative study has been made of human precipitating and co-precipitating anti-tetanus toxin antibodies. IgO co-precipitating antibody represented 10% of the total antibodies in the serum and had immunological and biological properties similar to those described for co-precipitating antibodies of other animal species. Human precipitating and co-precipitating anti- bodies had the same electrophoretic mobility and were localized in the same immunoglobulin fraction. By immunoprecipitation it was not possible to find antigenic differences between precipitating and co-precipitating antibodies. Both antibodies were localized in the IgG1 and IgG3 subclasses and neither were in the IgG4 subclass. Only the precipitating antibody can form insoluble complexes with antigen. Precipitating and co-precipitating antibodies agglutinated sensitized sheep red cells, however, only the precipitating antibody agglutinated human red cells. Eight to ten times more co-precipitating antibody was required to obtain a positive reaction in PCA. Precipitating antibody activated the complement system while co-precipitating antibody lacked this capacity. This difference in behaviour could not be attributed to localization of both antibodies in different IgO subclasses. Precipitating and co-precipitating antibodies were cytophilic. Only the former activated phagocytosis and increased clearance of antigen from the blood. These results are not surprising since co-precipitating antibody does not fix complement. Competition between human precipitating and co-precipitating antibodies in opsonization was analysed. In this test competition of both antibodies for the antigen depends on their respective amounts The K= 0.18 diminished to 005 when the ratio of pp:cop. antibody changed from 10:30 to 30:70. The fact that co-precipitating antibody was isolated from the sera of vertebrates other than man indicate that this antibody could possibly play a role in some immune mechanisms. Taking into account that in previous papers we have demonstrated that co-precipitating antibody functions as a molecule with one combining site of high affinity and one of low affinity, we have proposed that this antibody could function univalently and blocks the antigen. This could facilitate chronic parasitic, bacterial and viral infections, tumour growth and other chronic infections. [ABSTRACT FROM AUTHOR]

In this paper, a small-angle neutron scattering study of γII-crystallins near their isoelectric point is presented. The experiments were carried out using protein concentrations of 5.7-85.7 mg/ml at temperatures in the range 11-60°C. The experimental data were analyzed using an ellipsoidal model for intraparticle correlations and the mean spherical approximation for interparticle correlations. Our studies revealed that γII-crystallins have a thick hydration layer, which is possibly due to the special arrangement of polar and ionic groups on their surface. The temperature scan shows that, as a result of relatively strong attractive forces, clusters of two, three, or higher oligomers are present below 20°C. Our results suggest that protein clusters, with a distinctive hydration layer, form a protein-rich phase that separates from a protein-lean phase as the temperature is decreased below some threshold value. [ABSTRACT FROM AUTHOR]

Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Ash300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S.A., Hemling, M.E., Folena-Wasserman, G., Sweet, R. W., Anumula, K., Barr, J. R., Huddleston, M.J. & Taylor, P. (1989) J. Biol. Chem. 264, 21286–21295]. Among oligosaccharides released from sCD4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase-treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase-treated oligosaccharides of sCD4 as follows: [This symbol cannot be presented in ASCII format] where ± indicates residues present on only a proportion of chains. The spectrum of oligosaccharide structures released from each glycosylation site was assessed as being similar to that of total oligosaccharides on the basis of their chromatographic profiles on the lectin columns and on Bio-Gel P-4. [ABSTRACT FROM AUTHOR]

The mitochondrial inner membrane anion channel catalyzes the electrophoretic transport of a wide variety of anions and is inhibited by matrix divalent cations and protons. In this paper, evidence is provided that mersalyl and p-chloromercuribenzene-sulfonate each interact with this uniporter at two distinct sites. Binding to site 1 causes a shift in the pH dependence of transport, characterized by a decrease in the pIC50 for protons from about 7.8 to about 7.3, and leads to substantial stimulation of transport in the physiological pH range. This effect is not reversed by addition of thiols such as thioglycolate. Binding of mersalyl and p-chloromercuribenzenesulfonate to site 2 inhibits the transport of most anions including Pi, citrate, malonate, sulfate and ferrocyanide. The transport of Cl- is inhibited about 60% by mersalyl, but is not inhibited by p-chloromercuribenzenesulfonate. These data suggest that inhibition is a steric effect dependent on the size of the anion and the size of the R group of the mercurial. This inhibition is reversed by thioglycolate. Dose/response curves indicate that mersalyl binds to site 1 as the dose increased from 7 to 13 nmol/mg, whereas it binds to site 2 as the dose is increased from 10 to 18 nmol/mg. Thus, at certain pH values both stimulatory and inhibitory phases can be seen in the same dose/ response curve. It is suggested that these sites may contain thiol groups and that physiological regulators may exist which can effect changes in activity of the inner membrane anion uniporter similar to those exerted by mercurials. [ABSTRACT FROM AUTHOR]

It has been concluded from circumstantial evidence obtained with HeLa cells in vivo that the phosphorylation of ribosomal protein $6 increases the affinity of 40S particles for mRNP [Duncan, R. and McConkey, E. H. (1982) Eur. J. Biochem. 123, 535-538; Thomas, G., Martin-Pérez, J., Siegmann, M. and Otto, A. M. (1982) Cell 30, 235-242]. This conclusion needs to be tested in vitro in a reinitiating cell-free translation system from growth-competent cells. We have prepared such a system from HeLa cells and have compared the capacity of homologous 40 S subunits of various degrees of phosphorylation to enter the existing polysome pool. The 40S subunits' degree of phosphorylation was manipulated by exposing aliquots of growth-stimulated HeLa cells to hyperthermia (see accompanying paper). 40S subunits from beat-shocked and control cells, despite differences in $6 phosphorylation level as verified by two-dimensional electrophoresis, did not differ with respect to their recruitment into the existing polysome fraction. Owing to the reinitiation activity of the translation system, assay times could be kept sufficiently short, to avoid any serious interference by the $6 phosphatase activities of the system. Our results suggest that increased $6 phosphorylation by itself is not sufficient to accelerate the participation of 40S subunits in protein synthesis. [ABSTRACT FROM AUTHOR]

A NADPH-cytochrome c reductase (called Fraction II) has been solubilized with Triton X-100 from porcine liver microsomes and purified. 1. Fraction II can reconstitute with a cytochrome P-450 fraction (called Fraction I) solubilized with Triton X-100 from porcine kidney cortex microsomes to exhibit laurate ω-hydroxylation activity. 2. When the purified Fraction II is subjected to disc electrophoresis on polyacrylamide gel in the presence of 0.1% Triton X-100, it shows a single hand. In contrast, on electrophoresis in the absence of Triton X-100, a polydisperse state is obtained. Fraction II is estimated to have a molecular weight of about 400000 by gel-filtration with detergent, and it does not contain iron. 3. Either gel filtration on a Sephadex G-100 column or treatment with trypsin at 4 °C results in a conversion of Fraction II to a form, which migrates at a much faster rate on disc gel electrophoresis than dose Fraction II. This fast-moving form (called Fraction IIf) is indistinguishable from either steapsin or trypsin-solubilized NADPH-cytochrome c reductase, judging from several criteria including electrophoretic and sedimentation behaviors, and molecular size. 4. Fraction IIf is reconstitutively inactive in the hydroxylation, unless supplemented with spinach ferredoxin-NADP reductase. Incubation of Fraction IIf with trypsin at 30 °C results in a remarkable enhancement in the reconstitution activity in the presence of ferredoxin-NADP reductase. The results in this paper along with an earlier report [11] suggest that Fraction II may contain two different electron carrier components, possibly participating in the electron transfer from NADPH to cytochrome P-450. [ABSTRACT FROM AUTHOR]

α-Lactalbumin was isolated from human milk in a yield of 1.8 mg/ml of milk. The purification procedure involved ammonium sulphate fractionation (30% to 80% saturation) and pH-4.0 precipitation, followed by gel filtration with Sephadex G-100. A final purification stage using DEAE-cellulose was necessary in some preparations. Peptides derived from the reduced, S-aminoethylated protein by treatment with cyanogen bromide and digestion of these CNBr fragments with trypain, chymotrypsin or thermolysin, were purified by gel filtration, ion exchange chromatography and high-voltage paper electrophoresis. From the sequences of these peptides it has proved possible to deduce unambiguously the complete primary structure of the protein. Comparison with bovine α-laotaIbumin shows an identity in 72% of the residues with a further 6% being chemically similar amino acids. The corresponding figures for the human α-Iactalbumin/human lysozyme comparison, are 39% and 12%, respectively. The significance of some of the amino acid replacements is discussed. [ABSTRACT FROM AUTHOR]

An immunoglobulin M from Waldenström's disease (IgM Ga) was hydrolysed by papain and a glycopeptide fraction was isolated by gel filtration on Sephadex G-100. This fraction was then successively resolved into fourteen glycopeptides by gel filtration on Sephadex G-50 and by preparative paper electrophoresis at pH 1.9. The amino acid and carbohydrate content of these glycopeptides was determined by ionexchange chromatography in an autoanalyzer. All of them contain aspartic acid and serine, but they are quite different from one another, in amino acid composition and sequence about, the carbohydrate-peptide linkage, especially with respect to histidine, tyrosine, threonine, valine, glutamic acid, lysine and proline. Furthermore, a number of glycopeptides contain only mannose and glucosamine, while others contain in addition, galactose, fucose, and N-acetylneuraminic acid, in variable amounts. These results suggest the presence of several linkage sites of the oligosaccharide chains on the H-chain and the true heterogeneity of these oligosaccharide chains. The peptide sequence of a few glyeopeptides was reported, especially the following: r-Asn(carbohydrates)-Ser-Thr, also present, in the Fc fragment of Immunoglobulin G. [ABSTRACT FROM AUTHOR]

In order to determine the primary structure of the bovine β-caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr). The tryptic hydrolyzate was separated by column chromatography on Dowex-50. After additional purifications, 13 peptides numbered Ti to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino-acid analyses were carried out on all these peptides. Peptide T1 contain 2 Arginyl residues, one of which is NH2-terminal. This peptide represents the NH2-terminal end of the β-casein. Its composition is identical to that reposed by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β-casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2terminal. The COOH-terminal peptide was identified with peptide T4, devoid of basic aminoacids. The 14 tryptic peptides account for all the residues of the β-casein A2 when compared with the already published amino-acid analyses of the whole protein [2, 3]. Several other peptides, originating from incomplete or non-specific cleavages, were also obtained. Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β-casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G-50. The seventh peptide was obtained by chromatography on Dowex-50 of the whole CNBr-digest. After additional purifications, the ammo-acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2terminal end of β-casein. The COOH-terminal end of β-casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr-peptides account for all of the amino-acid residues of β-casein A2. β-casein A2 has 208 amino-acid residues. Its molecular weight is very close to 24 000. [ABSTRACT FROM AUTHOR]

Thiolase was purified 600–700 fold from a crude extract of pig heart by use of differential adsorption on Al(OH)3 gel, ammonium sulfate fractionation and ion exchange chromatography on DEAE­Sephadex and phosphorylated cellulose. The enzyme preparation is homogenous by the following criteria: sedimentation analysis and electrophoresis on polyacrylamide gel and on cellulose acetate membranes. The isoelectric poit of the protein was found to be approximately pH 7.2–7.3. The enzyme crystallized from ammonium sulfate solutions as thin plates of irregular shape. Crystalline thiolase stored for 18 months at –10° did not show any loss of activity. The thiolytic activity and the acyl transferase activity of the enzyme could be demonstrated separately. The thiolytic activity was demontrated by the specific incorporation of 14C from [1 ­14C]­acetyl­CoA into the carboxyl position of unlabelled acetoacetyl­CoA on incubation wtih the enzyme. The acyl transferase activity eas studied using either [1­14C]­CoA and patethein as substrates or 14C­labelled CoA and acyl­CoA derivatives of varius chain length. A remarkable specificity was found for the transfer of the acetyl group. Thiolase activity and acyl transferase activity of the enzyme were diminished with the same kinetics during heat treatment or incubation with N ­ethylmaleeimide, ρ­cholomercuribenzoate or iodoacetamide. this indicates that both activities belong to the same protein. Inhibition of thiolase and acyltranferase activities by iodoacetamide (5 x 10­5 could be prevented by preicubation of the enzyme with acetoacetyl­CoA or acetyl­CoA. The acyl transfer is the rate determining step when the thiolase reaction operates in the direction of chain cleavage. Incubation of thiolase with acetyl­Coa results in binding of acetyl groups to the protein. The uptake of acetyl groups is dependent upon the specific activity of the enzyme and corresponds to 3 acetyl residues per molecule of protein (molecular weight 170,000) for the highest enzymatic activity obtained. both incorporation of acetyl groups into the enzyme and thiolase activity are inhibited to the same extent by iodoacetamide. the incorportion of radioactivity into the enztme during incubation with iodo­[1­14C]­acetamide can be divided into two different reactions: a fast incorporation with accompanying loss of enzymatic and a much slower process which continues after complete inactivation. From the initial rate of incorporation, it was calculaetd that incorporation of 3.1 carbamoylmethyl residues per molecule of protein would completely inhibit the enzyme withthe highest obtained activity. this ration cmpares well with the results obtained by acetyl binding studies and indicates a minimum of 3 active sites per molecule of enzyme. Enzyme labelled with iodo­[1­14C]acetamide was hydrolyzed with 6 N HCl. Approximately 95% of the radioactivity in the hydrolysate was found to coincide with S­carboxymethyl­L­cystine on electrophoresis and paper chromatography. Repeated crystallization with authentic carboxymethyl­cystine did not result in a decrease of specific radioactivity. [ABSTRACT FROM AUTHOR]

An investigation has been made of the acid soluble protein fractions of wool that has been reduced with benzylmercaptane and treated with methyliodide. When this modified wool was shaken in dilute formic acid (50 v/v) at room temperature for 1 hour, 7% of a protein material was dissolved. After dialysis and subsequent lyophylisation the protein material was isolated, and shown to be heterogeneous by paper electrophoresis. Fractionation of this protein on Sephadex G-75 in dilute acetic acid gave more than six components of relatively high content of tyrosine and phenylalanine. One of these fractions, obtained in good yield, was purified by rechromatography. For further separation the protein fraction was subjected to ion exchange chromatography on Dowex 1 (×2) columns. By stepwise elution with buffers of decreasing pH-values, containing acetic acid and 8 M urea, four subfractions were obtained. The amino acid compositions of reduced wool, of the protein obtained by formic acid extraction, of the fraction after gel filtration on Sephadex G-75 and of one subfraction on Dowex 1 (×2) are given. In all three fractions an increase in glycine, tyrosine and phenylalanine content was observed with relatively low amounts of basic and acidic amino acids. The subfraction obtained after the final step of separation on Dowex 1 (×2) was further characterised by endgroup determinations. Dnp­glycine was the only N-terminal amino acid with traces of Dnp­alanine as contaminant. Hydrazinolysis resulted in tyrosine on the C-terminus. After digestion of the subfraction by trypsin and chymotrypsin a number of peptides with different chain lengths was obtained, indicating that, tyrosyl residues had accumulated and that larger arginyl­peptides were present. Sedimentation measurements by ultracentrifugation indicated that the subfraction had a weight average of apparent molecular weight in the range of 9,000 to 13,000. This is in agreement with the molecular weight of 16,400 derived from the amount of Dnp­glycine and the calculated one from the amino acid composition. The analytical results show that the subfraction represents a single polypeptide chain containing only one N-terminal residue and about hundred aminoacids of which about fifty are glycine, tyrosine and phenylalanine. [ABSTRACT FROM AUTHOR]

The pure lipopolysaccharide of Shigella sonnet phase II was submitted to acid hydrolysis (0.1 N H2SO4, 20 min, 100°), and from the hydrolyzate the specific fragment was isolated and purified. The analysis of the specific fragment showed that it contained about 70% sugar; the major constituents were glucose, galactose and heptose in the appropriate proportion 3:2:2.2. 2-Keto-3-deoxyoctonate (3.2%), glucosamine (3.4%) and O-phosphorylethanolamine (6.0%) were also found. The molecular weight of the specific fragment estimated by gel filtration ranged between 4,500-5,000. Electrophoretic experiments indicated its acid character: the isoelectric point was near 2.9. [ABSTRACT FROM AUTHOR]

Rabbit kidney tissue contains antigens which are tissue-specific and species restricted, as well as other antigens which are shared by different organs of the animal. The stability of these antigens was investigated as a function of temperature, in order to explore the possibility of thermal fractionation. It was observed that one rabbit kidney-specific autoantigen was destroyed at 56° and another at 65°. A third antigen, which is restricted to the rabbit species but is non-tissue specific, was destroyed at 72°. Ultracentrifugal analysis of the saline extract at different concentrations showed the presence of several components, whose extrapolated values at zero concentration were found to be 4.2S, 6.2S, 10S, 19S, and 80S. The first two components were the most prominent. The rabbit kidney-specific autoantigens were fractionated by a first step of salting out with ammonium sulphate at 4°. The fraction that precipitated in the range from 2.00 to 3.00 M retained most of the antigenic activity. As this fraction was quite impure, it was chromatographed through DEAE-cellulose, then processed through gel filtration columns, using Sephadex G-100 and G-200, and finally purified in agar zone electrophoresis. Two antigens were isolated and both shared similar antigenic characteristics. However, they had slight differences regarding their physicochemical properties. Sedimentation coefficients of 4.6S and 4.8S were obtained for them. Both antigens possessed a slow electrophoretic mobility, similar to that of the β-globulins. The antigen with higher sedimentation value was slightly faster electrophoretically than the other. [ABSTRACT FROM AUTHOR]

Elucidation of the molecular mechanism of silver nanoparticle (AgNP) synthesis is necessary to control nanoparticle size, shape, and monodispersity. In this study, the mechanism of AgNP formation by Neurospora intermedia was investigated. The higher production rate of AgNP formation using a culture supernatant heat‐treated at 100° and 121°C relative to that with an un‐treated culture supernatant indicated that the native form of the molecular species is not essential. The effect of the protein molecular weight (MW) on the nanoparticle size distribution and average size was studied by means of ultraviolet–visible spectroscopy and dynamic light scattering. Using un‐treated and concentrated cell‐free filtrate passed through 10 and 20 kDa cut‐off filters led to the production of AgNPs with average sizes of 25, 30, and 34 nm, respectively. Also, using the permeate fraction of cell‐free filtrate passed through a 100 kDa cut‐off filter led to the formation of the smallest nanoparticles with the narrowest size distribution (average size of 16 nm and polydispersity index of 0.18). Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of the fungal extracellular proteins showed two notable bands with the MWs of 15 and 23 kDa that are involved in the reduction and stabilisation of the nanoparticles, respectively. [ABSTRACT FROM AUTHOR]

Protein analysis is an important approach for disease diagnosis, in which sample pretreatment is an essential step since protein samples are often complex and many protein biomarkers are of low abundance. Here, given the good openness and light transmission of liquid plasticine (LP), which is a liquid entity formed by SiO2 nanoparticles and encapsulated aqueous solution, we developed a LP‐based field‐amplified sample stacking (FASS) system for protein enrichment. The system was composed of a LP container, a sample solution and a Tris‐HCl solution containing hydroxyethyl cellulose (HEC). The system design, mechanism investigation, optimization of experimental parameters and characterization of LP‐FASS performance for protein enrichment were well studied. Under the optimized experimental conditions of 1 % HEC, 100 mm Tris‐HCl and 100 V in the LP‐FASS system, a 40–80 times enrichment of proteins was obtained in 40 min using bovine hemoglobin (BHb) as the model protein using the constructed LP‐FASS system. The simultaneous enrichment of multiple proteins (phycocyanin, BHb and cytochrome C) was also realized using the system. The LP‐FASS system can serve as a new platform for protein enrichment which is easy to be combined with online and offline detections. [ABSTRACT FROM AUTHOR]

This study aims to produce electrospun cellulose acetate (CA) membrane as the alternative supporting medium for a separation of crude polysaccharides by electrophoresis and a screening of their antibacterial activity. Among the tested conditions of fabrication, electrospun CA membrane at 57% porosity showed the best separation of each polysaccharide from the standard mixture and the crude extract of Aloe vera via electrophoresis. As compared with the commercial CA membrane, the produced electrospun CA membrane demonstrated more separated spots of polysaccharides. The antibacterial activity of the electrophoretic polysaccharide was also determined against Escherichia coli and Staphylococcus aureus as the inhibition zone after the bacterial culture agar was overlaid on the membrane and incubated for 24 h. The results of this study suggested the potential application of electrospun CA membrane combining with electrophoresis as a simple method for separating crude polysaccharides and screening for their antibacterial activity. [ABSTRACT FROM AUTHOR]