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In multidimensional separations, two or more independent separation methods are coupled in an effort to resolve complex mixtures. The displacement mechanisms of each method should be orthogonal, such that little correlation exists between the retention of compounds in each dimension. When multiple orthogonal separation methods are coupled such that all sample components are subjected to complete analysis on all dimensions, the method is considered “comprehensive”. The primary advantage of comprehensive multidimensional separations over their one-dimensional counterparts is the potential for dramatically enhanced resolution. High resolving power can be achieved because the peak capacity of a comprehensive multidimensional separation is roughly equal to the product of the individual peak capacities of each dimension. In this review, the theory and instrumentation of two-dimensional liquid chromatography (LC-LC) and liquid chromatography-capillary electrophoresis (LC-CE) separations are discussed. Some applications of these techniques to the separation of biological molecules are highlighted. Future directions for the development of multidimensional separations are also considered. [ABSTRACT FROM AUTHOR]

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37°C, respectively). The metal ions Cu2+ and Fe3+ (at a concentration of 5 mmol liter-1) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a Km of 0.0953 mmol liter-1 and a Vmax of 86.27 mmol liter-1 min-1 mg-1 of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-α-L-arabinofuranosyl)-(1→3)-O-βD-xylopyranosyl-(1→4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase. [ABSTRACT FROM AUTHOR]

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-aibumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65°C (D65°C values; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D65°C value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance. [ABSTRACT FROM AUTHOR]

Reversed-phase thin-layer chromatography plates can be effectively used for the separation of metal ions in low concentrations by classical low-voltage electrophoresis. Electropherograms of the ionic composition of hydrochloric and perchloric acid solutions of rhodium(III) and iridium(IV) were obtained. These electropherograms are comparable with the results of capillary electrophoresis. Special features of electrophoresis on reversed-phase plates were noted. [ABSTRACT FROM AUTHOR]

The positional isomers of nitro-, amino-, chloro-, and hydroxybenzoic acids were separated by capillary electrophoresis. The introduction of 1 to 10 mM of natural α- and β-cyclodextrins into the background electrolyte significantly enhanced the resolution of the capillary electrophoresis system in its zone version, while the isomers were not separated in the absence of cyclodextrins (CDs). Additives of both individual CDs and their mixtures were used. Micellar electrokinetic chromatography with or without CD additives was used for the separation of analytes along with capillary zone electrophoresis. [ABSTRACT FROM AUTHOR]

Background: Many antiangiogenic molecules are proteolytically cleaved from larger plasma proteins. For example, plasminogen activators cleave plasminogen into plasmin, and plasmin is converted into angiostatin in the presence of sulfhydryl donors. We thus investigated whether the antiangiogenic activity in plasma could be increased by treatment with recombinant tissue plasminogen activator (rt-PA) and the sulfhydryl donor captopril.Methods: Human plasma was treated with rt-PA (10 micro g/mL) and/or captopril (1 micro M). Angiogenesis was measured in vitro by human endothelial cell tube formation and endothelial cell proliferation and in vivo in mice with the Matrigel plug assay. Angiostatin was removed from treated plasma by affinity chromatography, immunoprecipitation, or ion-exchange chromatography, and the antiangiogenic activity of the depleted plasma was assessed by tube formation. Three cancer patients were treated with rt-PA and captopril, and their pretreatment and post-treatment plasmas were tested for antiangiogenic activity in vitro.Results: Angiogenesis in vitro was stimulated by untreated plasma and inhibited by plasma that had been treated with rt-PA and captopril but was not affected by treatment with rt-PA and/or captopril alone. In vivo angiogenesis in Matrigel plugs was substantially lower in mice treated with rt-PA and captopril than in untreated control mice. Antiangiogenic activity in treated plasma was largely retained after angiostatin was removed: treated plasma inhibited angiogenesis by 64.3% (95% confidence interval [CI] = 46.4% to 82.2%), relative to untreated plasma, and treated plasma depleted of angiostatin by affinity chromatography or immunoprecipitation inhibited angiogenesis by 65.1% (95% CI = 53.8% to 76.4%) or 63.7% (95% CI = 50.9% to 76.5%), respectively. Antiangiogenic activity of plasma from three cancer patients was higher after treatment with rt-PA and captopril than before such treatment.Conclusion: Treatment with rt-PA and captopril induced antiangiogenic activity in vitro and in vivo that appears to be independent of angiostatin. [ABSTRACT FROM AUTHOR]

An endo-β-1,4-glucanase (EG) was purified from ripe strawberry (Fragaria × ananassa Duch.) fruit using cellulose affinity chromatography. The purified enzyme gave a single protein band of 54 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this protein showed strong homology with the proteins encoded by recently identified EG genes from different strawberry cultivars and from Arabidopsis, pepper and tomato. The enzyme specifically cleaved the β-1,4-glucosyl linkages of xyloglucan but was unable to hydrolyze those of insoluble cellulose. The pH optimum and Km of the enzyme against the artificial substrate carboxymethylcellulose (CMC) were pH 5.0–7.0 and 1.3 mg ml–1, respectively. To assess the role of the Cel1 enzyme in fruit softening a cDNA of the corresponding fruit-specific and ripening-enhanced strawberry gene, cel1, was used to down-regulate cel1 gene expression in transgenic strawberry plants. In several primary transformants, cel1 mRNAwas strongly suppressed in ripe fruit. However, the EG activity and firmness of these fruit were indistinguishable from those of control fruit. The expression of a second gene, cel2, encoding a different strawberry EG was unaltered in the fruits of these transformants. The presence of the cel2 transcript in transgenic plants may have prevented the specific down-regulation of cel1 from revealing its role in fruit softening. [ABSTRACT FROM AUTHOR]

Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. `Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on `Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [3H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeat-units of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins. [ABSTRACT FROM AUTHOR]

The aim of the present study was to assess the antiatherogenic properties of Pinus pinaster (maritime pine) seed oil. To this end, the effects of P. pinaster oil supplementation on lipoprotein levels and atherosclerotic lesions were compared to those of lard or sunflower oil in apolipoprotein E-deficient mice. Plasma total cholesterol (P < 0.0001) and VLDL + intermediary density lipoprotein (IDL)-cholesterol (P < 0.0001) levels were lower in mice fed P. pinaster and sunflower oil than in those fed the lard diet. In contrast, triglycerides (P < 0.0001) and VLDL + IDL-triglycerides (P < 0.0001) levels were higher in mice fed P. pinaster oil than sunflower oil or lard. The VLDL + IDL lipid composition of apolipoprotein E-deficient mice fed P. pinaster oil was intermediate between that of lard-fed transgenic mice and that of wild-type mice fed nonpurified diet. Using the Triton WR1339 method, the fractional catabolic rate of plasma triglycerides was found to be lower in mice fed P. pinaster oil (P < 0.0001) than sunflower oil or lard diet, suggesting a defective clearance of triglycerides in the P. pinaster group. Finally, the susceptibility of triglyceride-rich lipoproteins to in vitro lipoprotein lipase-mediated lipolysis was lower in the P. pinaster oil-fed group than in the lard-fed group. Despite the differences in VLDL + IDL level and lipid composition, the surface areas of aortic atherosclerotic lesions were not significantly different among mice fed P. pinaster, sunflower or lard diets. In conclusion, the results of the present study indicated that feeding P. pinaster oil had no better preventive effect on aortic atherosclerotic lesion extension in apolipoprotein E-deficient mice than other saturated or polyunsaturated fats. [ABSTRACT FROM AUTHOR]

Five fructan exohydrolase activities from Lolium rigidum Gaudin were separated and partly purified by a combination of salt precipitation, affinity chromatography, gel-filtration chromatography, anion-exchange chromatography and isoelectric focusing. The activities were identified by incubating enzyme fractions with fructan from both Lolium rigidum and Cichorium intybus. On the basis of activities when (6,6,6)-kestopentaose and (1,1,1)-kestopentaose were used as substrates, it was concluded that three of the activities hydrolyzed β-2,6-glycosidic linkages faster than β-2,1-glycosidic linkages and two activities hydrolyzed β-2,1-glycosidic linkages faster than β-2,6-glycosidic linkages. Fructan exohydrolases that hydrolyze β-2,1-glycosidic linkages faster than β-2,6-glycosidic linkages, and fructan exohydrolases that hydrolyze β-2,6-glycusidic linkages faster than β-2,1-glycosidic linkages have not previously been identified together in a temperate grass. All fructan exohydrolases were inhibited markedly by sucrose. It is proposed that the classification of β-fructofuranosidases be reconsidered because EC 3 . 2 . 1 . 80 is presently used to designate all fructan exohydrolases irrespective of the rates at which they hydrolyze β-2,1- or β-2,6-glycosidic linkages in fructans. [ABSTRACT FROM AUTHOR]

Thyroglobulin antibodies in the sera from 31 patients with a variety of disorders were studied by isoelectric focusing. Only one gave a spectrotype indicative of a monoclonal response. the other 30 giving spectrotypes characteristic of polyclonal responses. There was evidence of clonal dominance in some of the sera and each gave a different spectrotype. Light chains were prepared from five thyroglobulin antibodies purified by affinity chromatography. There was no restriction in the speclrotypes when compared with light chains prepared from normal immunoglohulin. [ABSTRACT FROM AUTHOR]

Oral contraceptives (OC) can induce in some women, scrum immune complexes precipitated in 25% saturated (NH4)2SO4, especially in the case of a thrombotic complication. In this work, the presence of antiethinyloestradiol (anti-EE) antibodies in the complexes was investigated. Binding of ethinyloestradiol-³H (EE-³H), either by (NH4)2SO4, precipitate directly, or in equilibrium dialysis experiments with the anti-EE gamma-globulin isolated through a purification-activation method combining dilution chromatography and affinity chromatography, was measured. The purified proteins were identified by immunoprecipitation and isoelectrophoresis. Twelve sera from OC users and three from women who had never used them were tested. Eleven of the twelve users had serum immune complexes. Normal human gamma-globulin pools and gamma-globulins prepared from a rabbit immunized with EE, were used as negative and positive controls. Results demonstrated that serum complexes precipitated in 25% saturated ammonium sulphate contained EE-³H binding gamma-globulins. For seven out of eleven cases, the association constant Ka was calculated and varied from 4 × 105 M-1 to 2.6 × 107 M-1, and the valence from 1.4 to 2, a result consistent with an IgG antibody activity. These anti-EE antibodies were found in the serum complexes from women on OC with or without thrombosis. However their amount seemed to be greater in the cases with thrombosis. In the serum of women who had never used the pill, in the only OC user who had no serum complexes in 25% saturated ammonium sulphate, and in the pool of normal human gammaglobulins, no anti-EE activity was detected. [ABSTRACT FROM AUTHOR]

Twelve-day-old rats were fed a mixture of fast and slow chromato-graphically prepared fractions of bovine IgG, labelled with 125I and 131I. The pooled young rat sera were submitted to analysis by electrofocusing. The results show a wide variation of concentration quotients between the different pI peaks of IgG, with the more basic fractions being transmitted more readily in both fractions. One slow IgG peak (pI 7.8) in particular is transmitted very readily. These findings reinforce the concept of selectivity in transmission. The possible use of the young rat gut as a test for protein integrity is discussed. [ABSTRACT FROM AUTHOR]

Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electrondense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or H4+ concentration and reinvestigated the nature of the electron-dense bodies. Energy-dispersive X-ray analysis of the granules in the cells showed that they contain large amount of P, Mg, and K. Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates. 31P-NMR spectra also lacked the peaks in the -22-ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron-dense bodies of D. gigas did not consist not consist of polyphosphates. Using multinuclear NMR spectroscopy we showed that the electron-dense bodies of Dl. Gigan contained a novel metabolite which was identified as α-glucose 1,2,3,4,6-pentakis (disphosphate). [ABSTRACT FROM AUTHOR]

We present the in vitro expression and purification of N-terminal fragments of human perlecan in insect cells. Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus expression vectors (BEVs) encoding amino acids 1–196 (domain I), 1–404 (domain I+11a) and 1–506 (domain I+IIab). The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restriction enzyme analysis and Southern blotting. Northern hybridization and metabolic labeling with [35S]methionine showed that expression of the perlecan-(I–404) and the -(1–506)- peptide was successful, but in the case of tile perlecan-(1–196)-peptide no recombinant protein was produced. lmmunoblotting sbowed that both the (l–404)-peptide and (1–506)-peptide are recognized by 95J 10, a monoclonal antibody that was previously raised against perlecan-(24–404)-peptide expressed in Escherichia coli Gel permeation and anion-exchange chromatograhy were applied to purify the recombinant proteins. Glycosaminoglycans were demonstrated to be present. Deglycosylation with chondroitinase ABC showed that the perlecan-(1–404) peptide was glycosylated with chondroitin sulfate residues. Consistent with these results, glycosaminoglycans isolated from the perlecan-(1–404)-peptide were identified as chondroitin sulfate by agarose gel electrophoresis. Furthermore the perlecan (1–404)- peptide showed affinity to immobilized basic fibroblast growth factor. The availability of baculovirus- derived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future. Kewords: perlecan; structure; baculovirus expression; chondroitin sulfate; basic fibroblast-growth factor [ABSTRACT FROM AUTHOR]

A new sulfur-rich basic polypeptide. so called ω-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified ω-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis. The complete primary structure of ω-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteotytic cleavage. ω-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by elcctrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues. and has strong sequence identity (66%) with the sorghum SIα1 α-amylase inhibitor. ω-hordothionin. like γ-hordothionin. exhibited translation inhibitory activity on both cukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to γ-hordothiomn, ω-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare. ;γ-hordothionin also inhibited the α-amylase activity from human saliva, while ω-hordothionin and the other different genetic variants of thionins, α- hordothionin and β-hordothionin. failed to show any inhibitory effect. [ABSTRACT FROM AUTHOR]

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur-based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine-resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (CH2OH2H2pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high-level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations m positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs thru are conserved across all other known H2Pte synthase molecules. The amino acid alteration al residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines. [ABSTRACT FROM AUTHOR]

1. H+-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53000 and 48000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H+-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H+-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H+-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive. [ABSTRACT FROM AUTHOR]

The major phosphoprotein synthesized by cultured chicken embryo osteoblasts had a molecular mass of approximately 66 kDa. The 32P label on the protein was cleaved by acid phosphatase treatment and O- [32P]phosphoserine and O-[32p]phosphothreonine could be identified after partial acid hydrolysis. The phosphoprotein contributed approximately 2.0% of the total protein synthesized by osteoblasts and was shown to be secreted, as shown by its presence in the culture media. Glycosylation was demonstrated by the fact that it could be labelled with [3H]galactosamine. The major ≈ 66-kDa phosphoprotein was resolved by isoelectric focusing into three major variants with pI values ranging over 3.7–3.9; all three forms appear to be the result of variation in the extent of protein phosphorylation. An identical ≈ 66-kDa phosphoprotein could be extracted from chicken bones which had both the same range of pI values and an identical elution position following DEAE- Sephacel chromatography. Analysis of the protein isolated from bone demonstrated the presence of sialic acid and, while amino-terminal sequence analysis and internal tryptic fragment sequence analysis of about 25% of the protein revealed little similarity to the rat phosphoprotein osteopontin, a conserved nine-residue sequence spanning the Arg-Gly-Asp cell-binding site of the rat protein osteopontin, was identified in the ≈ 66-kDa chicken protein. Peptide mapping with Staphylococcus aureus V8 protease of the in vivo protein compared to the in vitro synthesized protein demonstrated identical peptide fingerprints. The two proteins also had comparable amino acid compo- sitions. Several smaller-molecular-mass phosphoproteins ranging in size over about 55–29 kDa were also observed in the HCl extracts of bone. Peptide mapping of these species demonstrated that the ≈ 66-kDa, ≈ 55-kDa, and ≈ 45-kDa species had a common core of peptide fragments. Pulse/chase experiments in culture revealed no evidence for a defined pathway of intracellular proteolysis associated with the ≈ 66-kDa species since this phosphoprotein remained the prevalent species after a 24-h chase. Because of the predominant association of all the smaller-molecular-mass forms with the cell layer and an absence of a quantitative conversion to any of the smaller forms over a 24-h chase, these results suggested that the lower-molecular-mass species were not the result of proteolytic processing during synthesis or secretion, but rather represent proteolysis of the ≈ 66-kDa component in the extracellular matrix. In summary, these results demonstrate that cultured osteoblasts synthesize a major 66-kDa phosphoprotein that is identical to the major HCl-extractable phosphoprotein present in postnatal chick bone. [ABSTRACT FROM AUTHOR]

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pl 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4), These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, Mr could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase. [ABSTRACT FROM AUTHOR]

A human malignant glioma cell line, U-343 MGa Cl 2:6, has previously been shown to secrete platelet-derived-growth-factor(PDGF)-like activity [Nistér, M., Heldin, C.-H., Wasteson, Å. and Westermark, B. (1984) Proc. Natl Acad. Sci. USA 81, 926-930]. We report here that this activity consists of three different molecules separable by reversed-phase chromatography and immobilized-metal-ion affinity chromatography. HPLC reversed-phase chromatography resolved two peaks of activity, which were denoted glioma-derived growth factor-I (GDGF-I) and GDGF-II. GDGF-I was purified to >90% purity; in SDS gel electrophoresis, it appeared as a 31-kDa component which by reduction was converted to 17 kDa. Immunoprecipitation of radiolabeled, reduced and alkylated GDGF-I with antisera made against peptides from the A and B chains of PDGF, gave a specific signal only with antiserum against the A chain. Furthermore, when reduced and alkylated GDGF-I was analyzed by reversed-phase HPLC, it eluted at the position of PDGF A chains. We conclude that GDGF-I is a homodimer of a polypeptide similar to the A chain of PGDF. ODOF-II was found to have higher mitogenic activity than GDGF-I. Analysis by immunoprecipitation with PDGF-chain-specific antisera revealed that GDGF-II contained a polypeptide similar to the B chain of PDGF. Immobilized-metal-ion affinity chromatography revealed that 95% of the mitogenic activity of GDGF-II consisted of a heterodimer of one A and one B chain, whereas 5% consisted of a B-chain homodimer. Thus, U-343 MGa Cl 2:6 cells secrete all three possible dimeric forms of PDGF. [ABSTRACT FROM AUTHOR]

A new acidic form of glutathione S-transferase (OST, p1 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (Mr or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the Mt in isoelectric point, activity towards I ,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn2. The activity was inhibited by sulfasalazine, an inhibitor of leukotrienc-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 μM and 3.5 mM respectively. A major OST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotrienc A4 was 100 gM, fourfold higher than that of GST-Yn1Yn1. These results suggest that mainly GST-Yn1Yn1 may be involved in leukotriene-C4 synthesis in rat brain. [ABSTRACT FROM AUTHOR]

Rabbit α1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Septiadex G-100, affinity chromatography on concanavalin-A - Sepharose and ion-exchange chromatography on DEAE-Sepbadex. Rabbit α1-microglobulin had a molecular mass of 25.6 kDa on SDS/ polyacrylamide gel etectrophoresis. α1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of α1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human α1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit α1-microglobulin. with a gap between each band of 2.6 - 2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig α1-microglobulin. Our results indicate that human α1-microglobulin is substituted with two N-linked oligosaccharides, white only one is attached to each of the other α1,-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the Nelinked oligosaccharides carry the immunosuppressive activity of α1-niicroglobulin. [ABSTRACT FROM AUTHOR]

Gamones 3 and 4 of the ciliate Euplotes octocarinatus were isolated and purified to chromatographic and electrophoretic homogeneity. They are secreted into the culture medium by cells of certain mating types and induce cells of other mating types to unite in pairs and exchange gametic nuclei. The purified gamones are biologically active at concentrations as low as 0.1–1 pM. Both are polypeptides with unusually low pI values of approximately 3.2, and both have a carbohydrate content of less than 2%. Gamone 3 has an Mr of 18800 and is slightly smaller than gamone 4, which has an Mr of 23500. Gamone 3 was isolated from the starvation medium of cells homozygous for the mating type allele mt³ and gamone 4 was isolated from the medium of cells homozygous for the mating type allele mt4. Each of the two homozygous cell lines was found to secrete one gamone only. [ABSTRACT FROM AUTHOR]

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55 000. The Mr on gel filtration was 44–48 000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing &assymp;10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase. [ABSTRACT FROM AUTHOR]

The plasma and serum of humans and various animal species exert an actin-depolymerizing activity. Human actin-depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE-cellulose and blue-Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin-sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin-A-Sepharose column. Incubation of ethanol-fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90-kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes. [ABSTRACT FROM AUTHOR]

Analysis by crossed-immunoelectrophoresis of Paracoccus denitrificans membrane vesicles has shown that only one antigen stains for NADH dehydrogenase activity. This activity could be partially purified by a combination of gel filtration and ion-exchange chromatography of membrane vesicles that had been solubilised in the non-ionic detergent Nonidet P-40. From the limited number of precipitates observed after crossed immunoelectrophoresis of this partially purified preparation of NADH dehydrogenase it was possible to excise specifically part of the precipitate that stained for NADH dehydrogenase. Excised precipitates containing NADH dehydrogenase that had been radiolabelled by growth of cells in the presence of [35S]SO42- allowed the polypeptide composition of the enzyme to be determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by fluorography. Two subunits were identified with estimated relative molecular masses of 48 000 and 25 000. Subunits of similar molecular weight are found in the flavoprotein fragment of the NADH dehydrogenase of the mammalian mitochondrial respiratory chain. The latter has general similarities with the respiratory chain in the plasma membrane of P. denitrificans. [ABSTRACT FROM AUTHOR]

Seryl-tRNA synthetase was purified 1800-fold from bovine liver extract by ultracentrifugation at 150000 x g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, gel chromatography on Sephacryl S-300, adsorption chromatography on hydroxyapatite, affinity chromatography on blue-Sepharose and finally on M&asline;trex gel red A. The relative molecular mass, Mr, in the denatured state was estimated as 87000 by sodium dodecyl sulfate disc gel electrophoresis; in the active state the Mr was estimated as 170000 for the dimeric native enzyme (α2 type) by chromatography on Sephacryi S-300. The amino acid composition of the enzyme was determined. The Km values for ATP and serine were 0.49 mM and 30 μM, respectively. The Km values for tNAIGASer and tRNAcmCASer were 1.40 μM and 1.25 μM, respectively. Sequences common to the two isoaccepting tRNASer molecules are discussed in relation to the recognition mechanism of the purified seryl-tRNA synthetase. [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]

A novel killer toxin, encoded by a double-stranded linear DNA plasmid pGK 1-1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320000-fold from the culture broth of yeast. The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis. The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished. The isoelectric point was between 4.4 and 4.8. As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double-stranded RNA plasmid of Saccharomyces cerevisiae, the K. Lactis toxin was effective with sensitive strains of S. cerevisiae in a relatively wider pH range between 4 and 7; it was stable for several months at pH 6.0 when stored below -20 °C. In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K. lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA-Sepharose column and eluted by methyl α-D-mannoside. The growth inhibitory activity of K. lactis toxin was enhanced 2-35fold by the presence of 4-60% glycerol. [ABSTRACT FROM AUTHOR]

Patterns of alkaline phosphatase (AP)-binding proteins were observed in the alkaline pH range of 6.5-9.5 upon isoelectric focusing and blotting of serum from patients with inflammatory diseases. After isolation using affinity chromatography on protein A or immunoaffinity chromatography on AP coupled to cyanogen bromide (CNBr)-activated Sepharose, the AP-binding protein was identified as IgG on Western blots and in ELISA using human IgG-specific antibodies. It was shown that this IgG binds to AP from both calf (bovine) and human intestine. However, it binds neither lo the human liver-bone-kidney (LBK) isoform nor to bacterial AP. Moderate reaction was observed with human placental AP. Comparing patients with various diagnoses (n = 284), AP-binding antibodies were mainly found in severe bacterial infections. They were not detected in serum from healthy blood donors (n = 300). The presence of AP-binding IgG was independent of the infected organ and the bacterial species causing infection. This antibody may be useful for discriminating bacterial from viral infection and for indicating severe bacterial inflammation. [ABSTRACT FROM AUTHOR]

The Spectrotypic patterns of anti-Echinococcus granulosus and anti-F(ab')2 antibodies were analysed in 13 patients with different forms of hydatidosis by means of isoelectric focusing (IEF), Specific anti echinococcus antibodies and specific echinococcus antigens were obtained by crossed affinity chromatography between patient serum and cyst fluid, as previously described (D'Amelio et al., 1985). A wide spectrotype in the pattern of anti-Echinococcus antibodies was observed, with strong homologies among the different sera mainly at acid and neutral pH. One serum displayed faint, nearly in apparent bands. Conversely, the pattern of anti-F(ab')2 antibodies in the six patients in whom they were positive was limited but heterogeneous, ranging from monoclonal through oligoclonal to polyclonal. A predominant clone was identified in the patient with a polyclonal pattern. A correlation between qualitative IEF spectrotypic patterns and the quantitative results of immunoenzymatic assays was observed for both types of antibodies. [ABSTRACT FROM AUTHOR]

A component of Aspergillus fumigatus, Ag 7, previously identified as a major antigen for patients with allergic bronchopulmonary aspergillosis, has been isolated by gel filtration and further purified by affinity chromatography using monospecific antiserum. The antigen, which binds both specific IgG and IgE antibodies, was shown to be a high molecular weight, 150-200 kD, heat-stable glycoprotein, which binds to concanavalin A. suggesting the presence of α-D mannopyrannoside, or α-D glucopyrannoside and residues. On sodium dodecyl sulfate polyaerylamide gel electrophoresis (SDS-PAGE) it had a subunit of molecular weight 36 kD (with or without prior reduction), which retained antigenicity and allergenicity when tested with patients" sera. [ABSTRACT FROM AUTHOR]

5′-Nucleolidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin-A- U]trogel and 5′AMP-Sepharose 4B followed by AcA-54-Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross-linking of the native enzyme with direct hylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5′-Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane-bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane-bound enzyme showed a wide range of pH optimum (7.5–8.3). An Arrhenius plot of the membrane-bound enzyme shows a break at 28°C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+. [ABSTRACT FROM AUTHOR]

Up to seven early poxvirus-specific proteins have been isolated from vaccinia-WR-infected and cowpox-virus-infected chick embryo fibroblasts by affinity chromatography on native DNA-cellulose columns. The proteins have been characterized by one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by nonequilibrium pH-gradient electrophoresis. The molecular weights of the viral proteins were determined by comparison with proteins of known molecular weight and are comparable to several of the vaccinia-WR-specific DNA-binding proteins isolated previously from infected L-929 cells by Solosky J,M., Esteban M. and Holowczak J. A. [J. Virol. 25, 263-273 (1978)]. The viral proteins binding reversibly to native DNA have been classified as immediate early viral gene products. Synthesis of cowpox-virus-induced early DNA-binding proteins is inhibited in chick cells pretreated with homologous interferon at a concentration of 500-1000 units/ml. [ABSTRACT FROM AUTHOR]

DNA-dependent RNA polymerase III has been purified to homogeneity from the filamentous fungus Podospota comata. The enzyme was extracted at low ionic strength, separated from the polymerases I and II by DEAE-Sephadex chromatography and purified by heparin-Sepharose and phosphocellulose chromatography; 0.1–0.2 mg highly purified homogenous enzyme with a specific activity of 220 units/mg could be obtained from 2 kg wet mycelium. The subunit composition of the enzyme was determined after sodium dodecyl sulphate/ polyacrylamide gel electrophoresis: thirteen putative subunits of molecular weight 174000 (a), 129000 b). 87000 (c), 50000 (d), 39000 (e), 23500 (f), 21000 (g), 19000 (h), 17000 (i), 16500 (j), 13500 (k), 11000 (l) and 10000 (m) were identified. All of the polypeptide components of the enzyme are present in about integral stoichiometric amounts as judged by dye binding. The presence of subunit Mr = 87000 in a molar ratio 1:1 is necessary to obtain very active enzyme. Thirteen homologous subunits were observed in a preparation of RNA polymerase III from Podospora anserine, which is a related species. Only subunit i is different in the two species. [ABSTRACT FROM AUTHOR]

Preulbumin was purified from rat plasma by chromatography on Blue Sepharose CL-6B, followed by chromatography on concanavalin-A—Sepharose and preparative electrophoresis in polyacrylamide gel. The overall recovery was 20%. The purified prealbumin was homogeneous upon electrophoresis in detergent containing polyacrylamide gel and was used to raise a monospecific anti-serum in rabbits. During perfusion of rat liver, [14C]-leucine was incorporated into prealbumin secreted into the perfusion medium, suggesting that the liver was synthesizing prealbumin. The ratio of the rate of incorporation of leucine into prealbumin to that into total protein was 1.5%. 125I-prealbumin had a half-life of 29 h in the bloodstream of healthy Buffalo rats on a diet containing 20% protein. Whole body homogenates from healthy rats contained 6.2 mg prealbumin/100 g body weight. The rate of synthesis of prealbumin calculated from half-life and total body pool, was 3.6 mg prealbumin ×(100 g body weight)-1 × day-1. Two days after induction of inflammation by a subcutaneous injection of turpentine, both the concentration of prealbumin in the plasma and the amount of prealbumin in the whole body homogenates decreased to a minimum of about one third of normal. The relative rate of degradation of 125I-prealbumin did not change during inflammation. The rate of synthesis of prealbumin decreased considerably, or even ceased, during acute inflammation. [ABSTRACT FROM AUTHOR]

Rat liver microsomal glutathione 5-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the giutathione S-transferase accounts for 2.5 – 3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14000 on sodum dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127000 and contains three or four polypeptide chains and 112–134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione 5-transferase is an enzyme distinct from the cytosolic glutathione 5-transferases. [ABSTRACT FROM AUTHOR]

1. CM-cellulose chromatography of a fraction soluble in 5% perchloric acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which have been purified to electrophoretical homogeneity. 2. C1a1, C1a2 and C1b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophila D1 [Rodriguez Alfageme et al. (1980) Chromosoma, 78, 1-31] are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do non appreciably fold on increasing ionic strength. 3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2. [ABSTRACT FROM AUTHOR]

1. High-voltage electrophoresis and chromatography before and after reaction with 5-dimethylaminonapthalene-1-sulphonyl chloride have identified putrescine and spermidine in hydrolysates of cyst coat proteins from the protozoan Colpoda steinii. 2. Amounts present varied with putrescine up to 19.7 and spermidine up to 16.9 residues per 1000 amino acid residues. 3. The amines were not, in the main, removed by acid or alkaline extraction or by reprecipitation. They were present in hydrolysates of peptides isolated electrophoretically from acid-degraded coat protein. 4. Proteolysis of oxidized coat protein produced a soluble core polypeptide to which the major proportion of the amines were attached and which had a simple composition. It was composed almost entirely of glutamic acid or glutamine, glycine, serine and cysteic acid, these residues being present in the approximate ratio of 10:2:1:1. 5. When coat protein was treated with 5-dimethylaminoaphthalene-1-sulphonyl chloride and hydrolysed no fully substituted amines could be detected but putrescine with one group substituted and spermidine derivatives with one and two groups substituted were present. [ABSTRACT FROM AUTHOR]

High-molecular-weight (high-MT) kininogen was purified from horse plasma by chromatography on columns of DEAE-Sephadex A-50, CM-Sephadex C-50, p-chlorobenzylamine-Sepharose and Sephadex G-150. The yield was about 151 mg from 8.1 of fresh plasma. The purified material gave a single band on sodium dodecylsulfate/ polyacrylamide gel electrophoresis and a single precipitin line on immunodiffusion and immunoelctrophoresis. The molecular weight of horse high-MT kininogcn was estimated to be 78000 by dodecylsullate gel electrophoresis using the Ferguson plot. Its polypeptide content was determined to be 86% by amino acid analysis and there was a total of 581 amino acid residues/molecule of protein The kininogen contained a total of 13.9% carbohydrates, consisting of hexoses (7.8%), glucosamine (1.9%), galactosamine (0.6%) and sialic acid (3.6%). On incubation of horse high-MT, kininogen with bovine and horse plasma kallikreins, several fragments winch contained extremely high levels of histidine, were liberated, in addition to kinin. After the liberation of kinin and histidine-rich fragments, a protein free of kinin and its fragments was isolated. This protein consisted of two polypeptide chains, heavy chain and light chain, which are bridged by disullide bonds. The molecular weight and amino acid composition of the heavy chain anti the light chain from horse high-MT kininogen were very similar to those of the heavy and light chains from bovine high-MT kininogen. respectively. From these results, it was revealed that horse high-MT kininogen is quite similar to bovine high-MT kininogen in terms of their physicochemical and chemical properties, although they are immunologically distinguishable. [ABSTRACT FROM AUTHOR]

Three sialoglycoproteins (S[SUB1], S[SUB2], and S[SUB3]) have been isolated from normal human urine by ultrafiltration, zone electrophoresis, gel chromatography, and ion-exchange chromatography. The average yields per litre of urine were 0.39 mg (S[SUB1]), 0.40 mg (S[SUB2]), and 1.9 mg (S[SUB3]). The isolated substances were homogeneous on ultracentrifugation both at neutral and acid pH with sedimentation coefficients of 3.6S (S[SUB1]), 2.4S (S[SUB2]), and 0.93S (S[SUB3]). Equilibrium ultra centrifugation gave molecular weights of 77 900 (S[SUB1]), 37 000 (S[SUB2]), and 5300 (S[SUB3]). All three components were rich in carbohydrate (64 to 76%) and contained 28 to 35% of sialic acid. Alkaline borohydride degradation of component S3 yielded two sialylated oligosaccharides which were partially characterized. The origin of the isolated substances is unknown. The molecular size of S[SUB3] (Stokes' radius of 2.0 nm) is compatible with passage from the blood by glomerular filtration whereas the size of S[SUB1] (Stokes' radius of 6.5 nm) would suggest a renal origin. [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]

We have utilized [3H]cDNA · DNA annealing (using S1 endonuclease resistance as an assay) to detect the presence of sequences complementary to chick δ-crystallin mRNA in the genome of fish (salmon, herring), amphibians (frog, newt), reptiles (python, gekko, caiman), birds (quail, turkey, duck), and mammals (mouse, calf), and have compared several physical parameters of δ-crystallin of the reptiles and birds. The rate and extent of chick δ-crystallin [3H]cDNA · DNA annealing were comparable among the birds, and indicated that the δ-crystallin sequences are in the unique fraction of the genome. On the average, about one-fifth as much annealing took place with the reptiles as with the birds, and no annealing was observed with the fish, amphibians and mammals. The avian and reptilian δ-crystallins had subunit molecular weights near 50000 and native molecular weights near 200000, as judged by sodium dodecylsulfate/urea/polyacrylamide gel electrophoresis and agarose gel chromatography, respectively, indicating that the subunit structure of δ-crystallin has been largely conserved. Electrophoresis of the peptides generated by digestion with Staphylococcus aureus V8 protease indicated that the primary structures of the different reptilian and avian δ-crystallins were similar but not identical. The isoelectric points of the δ-crystallins ranged between pH 5 and 7, depending on the organism. In general, the reptilian δ-crystallins had the higher isoelectric points. [ABSTRACT FROM AUTHOR]

A protein with properties similar to apoprotein A-IV of rat high-density lipoproteins has been isolated from the lipoprotein fraction (e < 1.006 g/ml) of individual non-fasting hypertriglyceridaemic subjects. The Mr of human apolipoprotein A-IV determined by polyacrylamide-gel electrophoresis in sodium dodecylsulfate and by column chromatography in 6 M guanidine hydrochloride is 46000. The amino acid composition is significantly different from any previously characterised human apolipoprotein but resembles that of rat apolipoprotein A-IV. The N-terminal amino acid of human and rat apolipoprotein A-IV is glutamate or glutamine. The pI of the major human A-IV band in 6 M urea is &ap; 5.15. Immunochemically apolipoprotein A-IV is not identical with any of the well known human apolipoproteins. Human apolipoprotein A-IV is present in all fractions of e < 1.006 g/ml isolated by a single ultracentrifugal spin from non-fasting subjects, but is lost from lipoproteins of e < 1.006 g/ml upon recentrifugation. Irnmunoelectrophoretic studies show that most of human apolipoprotein A-IV is present in the fraction of e < > 1.21 g/ml and is unassociated with the major lipoprotein fractions in serum. Charge- shift electrophoresis however clearly demonstrates the hydrophobic character of human apolipoprotein A-IV. There is evidence that apolipoprotein A-IV is a constituent of chylomicrons that is removed from the particle surface when chylomicrons enter the plasma compartment. [ABSTRACT FROM AUTHOR]

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

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 µM, 18 µM, and 50 µM respectively. [ABSTRACT FROM AUTHOR]

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticoagulant activity and one of low anticoagulant activity by affinity chromatography on matrix-bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III. [ABSTRACT FROM AUTHOR]

The nuclear enzyme poly(ADP-ribose) polymerase has been purified about 9200-fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub-zero temperatures. Electrophoretic analysis under both denaturing and non-denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate-/polyacrylamide gel electrophoresis was 63500 and from gel filtration through columns of Sephadex G-100, 58000. The enzyme preparation was free from poly(ADP-ribose)-degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 μmol NAD+ incorporated × min-1 × mg-1 of protein at 37 °C. Amino-terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino-terminal group may be blocked. In the presence of histones, the Km for NAD+ was 23 μM. [ABSTRACT FROM AUTHOR]

1. A detergent-solubilized pig intestinal microvillus aminopeptidase (microsomal aminopeptidase, EC 3.4.11.2) was purified approximately 150 times with a yield of about 20% by a rapid and simple immunoadsorbent technique. The purified enzyme was shown to be homogeneous by crossed immunoelectrophoresis and by polyacrylamide gel electrophoresis in dodecyl sulphate. Furthermore it was essentially free from other known peptidases and disaccharidases. 2. By charge-shift crossed immunoelectrophoresis it was demonstrated that the hydrophilic form was continuously generated both during a more time-consuming purification procedure and during storage. The development of a rapid purification procedure allowed the preparation of an amphiphilic form free from the hydrophilic one, which could be stabilized by 0.5 mM HgCl2. 3. Intestinal aminopeptidase isolated from a pig having the pancreatic duct ligated three days before sacrifice migrated in polyacrylamide gel electrophoresis in dodecyl sulphate as a single polypeptide chain with a molecular weight of 162 000. In contrast, the enzyme from a pig with intact pancreatic duct when purified even in the presence of Kunitz trypsin inhibitor consisted of three polypeptide chains (molecular weights 162 000, 123 000 and 61 000). Trypsin treatment in vitro converted the large polypeptide chain into the two smaller ones. It is suggested that the membranebound enzyme after biosynthesis is modified by trypsin and/or chymotrypsin. 4. The conversion of the amphiphilic form to the hydrophilic one and the hydrolysis of the large polypeptide chain into the two smaller ones represented different reactions. [ABSTRACT FROM AUTHOR]