bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractionation and characterization of proteoglycans isolated from chondrocyte cell cultures
Authors: Björnsson Sven , Heinegȧrd Dick
Published: August 2015

Chondrocyte cultures were established from foetal bovine tracheal cartilage and maintained in Ham's F12 medium with or without 10% (v/v) foetal calf serum. The proteoglycans were isolated and characterized. (1) The proteoglycans from cultures both with and without serum distributed in associative or dissociative CsCl gradients like proteoglycans from cartilage tissue. (2) The amino acid composition, protein contents and glucosamine/galactosamine ratios were grossly identical with those of the tissue derived proteoglycans. (3) Sedimentation coefficients (s0) for the monomers were 21.0S and 22.7S from cultures without and with serum respectively. The s0 values obtained for aggregates were 72.3S and 93.2S respectively. The limiting viscosity numbers [η] were 248ml/g and 298ml/g respectively. These data corresponded well to those obtained for the tissue-derived proteoglycans. (4) The sizes of the core proteins and chondroitin sulphate chains respectively were the same for both types of cell-culture proteoglycans and similar to those of the tissue proteoglycans. Both the keratan sulphate-rich region and the hyaluronic acid-binding region were identified. The latter, however, was not resistant to limit digestion with trypsin, in contrast with the fragment derived from the bovine nasal cartilage. (5) About 70% of the cell-culture proteoglycans chromatographed in the void volume on a Sepharose 2B column, whereas reduced and alkylated samples (monomers) chromatographed completely included in the column. The two link proteins present in A1 preparations of cartilage proteoglycans were also present in A1 preparations of cell-culture proteoglycans. (6) A minor portion (10%) of the 35S-labelled proteoglycans in the cultures was associated with the cells. Reduced and alkylated samples were larger compared with the monomers in the medium, and chromatographed partly (25%) excluded on the Sepharose 2B column. A larger proportion (50%) of the non-reduced samples chromatographed in the void volume of the column.
Proline biosynthesis in Escherichia coli Stoichiometry and end-product identification of the reaction catalysed by glutamate semialdehyde dehydrogenase
Authors: Hayzer D J , Leisinger T
Published: August 2015

The stoichiometry of the oxidative phosphorylation of glutamic acid 5-semialdehyde by gamma-glutamyl phosphate reductase (glutamate semialdehyde dehydrogenase) has been established. Equimolar amounts of NADP+ and L-glutamic acid 5-semialdehyde are consumed and equimolar amounts of 5-oxiopyrroilidine-2-carboxylic acid and NADPH are formed. The end-product of the reaction is demonstrated to be 5-oxopyrrolidine-2-carboxylic acid, probably arising from the true end-product gamma-glutamyl phosphate.
A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage
Authors: Heinegård D , Paulsson M , Inerot S , Carlström C
Published: August 2015

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40-1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.
Isolation and analysis of human parathyrin in parathyroid tissue and plasma Use of reversed-phase liquid chromatography
Authors: Bennett H P J , Solomon S , Goltzman D
Published: August 2015

Reversed-phase liquid chromatography techniques have been used to extract and purify human parathyrin from parathyroid adenomas and to analyse the circulating forms of human parathyrin in plasma. Both the supernatant from tissue homogenates, and plasma were extracted with octadecylsilyl-silica (ODS-silica) in a batch procedure. Extracts were subjected to reversed-phase high-pressure liquid chromatography (h.p.l.c.) employing solvent systems composed of aqueous acetonitrile containing trifluoroacetic acid or heptafluorobutyric acid as hydrophobic ion-pairing reagents. The volatile solvents facilitated the radioimmunoassay, bioassay in vitro and amino acid analysis of column fractions and permitted monitoring for u.v. absorbance at 210nm. Isolated glandular parathyrin was found to be homogeneous by sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis, to have an amino acid composition conforming to that of human parathyrin-(1-84)-tetraoctacontapeptide and to be bioactive in both renal adenylate cyclase and cytochemical bioassays. ODS-silica extraction permitted examination of large plasms samples by reversed-phase h.p.l.c., facilitating the resolution of the various circulating molecular forms of parathyrin according to their hydrophobic character. Because of its rapidity, excellent recovery and high resolving power, the methodology utilized is uniquely suited to the purification and analysis of parathyrin in tissues and body fluids.
Molybdenum(V) e.p.r. signals obtained from xanthine oxidase on reduction with aldehyde substrates and with 2-amino-4-hydroxy-6-formylpteridine
Authors: Malthouse J P , Williams J W , Bray R C
Published: August 2015

2-Amino-4-hydroxy-6-formylpteridine, a known ‘slow’ substrate and inhibitor of xanthine oxidase, is unusual in that it gives rise under suitable conditions to all types of molybdenum(V) e.p.r. signals obtainable from the enzyme, namely Very Rapid, Rapid, Inhibited and Slow. The Very Rapid signal appears in a slightly modified form. The Inhibited signal, originally thought to be unique to reaction of methanol or of formaldehyde with xanthine oxidase, is now shown to be obtainable with several other aldehydes. These include, in addition to 2-amino-4-hydroxy-6-formylpteridine, acetaldehyde and glycoaldehyde. Parameters of the signals, obtained with the help of computer simulations, are presented. The appearance of Very Rapid and of Inhibited signals with these additional substrates may be of importance in elucidating the structure of the enzyme active centre. In agreement with previous work, the Very Rapid signal is attributed to an obligatory intermediate in turnover. On the other hand, the Inhibited signal is attributed to a side reaction, presumably inhibitory in nature, occurring during the catalytic process.
Cyclic nucleotide phosphodiesterase of rat pancreatic islets. Effects of Ca2+, calmodulin and trifluoperazine
Authors: Sugden M C , Ashcroft S J
Published: August 2015

Cyclic nucleotide phosphodiesterase activity towards cyclic AMP and cyclic GMP was studied in extracts of rat islets of Langerhans. Biphasic Eadie plots [Eadie (1942) J. Biol. Chem. 146, 85-93] were obtained with either substrate suggesting the presence of both ‘high’- and ‘low’-Km components. The apparent Km values were 6.2 +/- 0.5 (n = 8) microM and 103.4 +/- 13.5 (6) microM for cyclic AMP and 3.6 +/- 0.3 (12) microM and 61.4 +/- 7.5 (13) microM for cyclic GMP. With cyclic AMP as substrate, phosphodeisterase activity was increased by calmodulin and Ca2+ and decreased by trifluoperazine, a specific inhibitor of calmodulin. With cyclic GMP as substrate, phosphodiesterase activity was decreased by omission of Ca2+ or addition of trifluoperazine. Addition of exogenous calmodulin had no effect on activity. The data suggest that Ca2+ may influence the islet content of cyclic AMP and cyclic GMP via effects on calmodulin-dependent cyclic nucleotide phosphodiesterase(s).
Molecular and antigenic properties of cytochrome b5 from slow-muscle sarcoplasmic reticulum
Authors: Salviati G , Salvatori S , Betto R , Margreth A
Published: August 2015

NADH-cytochrome b5 reductase and cytochrome b5 associated with slow-muscle sarcoplasmic reticulum and liver microsomal fraction were identified with discrete protein bands of molecular weights 33000 and 16700 by polyacrylamide-gel electrophoresis. Purified detergent-extracted cytochrome b5 from muscle sarcoplasmic reticulum is indistinguishable from liver microsomal cytochrome b5 with respect to spectral properties, pI values and immunological reactivity with antibody to the liver cytochrome b5. Reaction of the antibody with membrane-bound cytochrome b5 inhibits the sarcoplasmic-reticulum NADH-cytochrome c reductase activity.
Primary structure of the ovine pituitary follitropin α-subunit
Authors: Sairam M R
Published: August 2015

All the tryptic peptides of reduced and aminoethylated alpha-subunit of ovine pituitary follitropin were isolated. From their their composition and partial sequence analysis of the N- and C-termini of the oxidized protein and reliance on homology with the sequence of lutropin alpha-subunit, an entire structure for the follitropin alpha-subunit has been proposed. The structure of the alpha-subunits from the two ovine hormones are identical.
Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen
Authors: McKenzie J L , Allen A K , Fabre J W
Published: August 2015

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.
Phenobarbital selectively modulates the glucagon-stimulated activity of adenylate cyclase by depressing the lipid phase separation occurring in the outer half of the bilayer of liver plasma membranes
Authors: Houslay M D , Dipple I , Gordon L M
Published: August 2015

The glucagon-stimulated (coupled) activity of rat liver plasma-membrane adenylate cyclase could be selectively modulated by the anionic drug phenobarbital, whereas the fluoride-stimulated (uncoupled) activity remained unaffected. It is suggested that the cationic drug phenobarbital preferentially interacts with the external half of the bilayer, as the negatively charged phospholipids are found at the cytosol-facing side. This results in a selective fluidization of the external half of the bilayer, leading to a depression in the high-temperature onset of the lipid phase transition (from 28 degree to 16 degree C) occurring there. This was detected both by e.s.r. analysis, using a fatty acid spin probe, and also by Arrhenius plots of glucagon-stimulated activity, where the enzyme forms a transmembrane complex with the receptor and is sensitive to the lipid environment of both halves of the bilayer. However, in the absence of hormone, adenylate cyclase only senses the lipid environment of the inner (cytosol) half of the bilayer. Thus its fluoride stimulated activity and Arrhenius plots of this activity remained unaffected by the presence of phenobarbital (less than 12 mM) in the assay. These results support the view that independent modulation of the fluidity or chemical constituents of each half of the bilayer can selectively affect the receptor-coupled and uncoupled activities of adenylate cyclase.
The molecular weight of Artemia ribosomes, as determined from their refractive-index increment and light-scattering intensity
Authors: Nieuwenhuysen Paul , De Voeght Frank , Clauwaert Julius
Published: August 2015

Cytoplasmic ribosomes were isolated from the cryptobiotic embryos of the brine shrimp Artemia salina. Measurements of their refractive-index increments and light-scattering intensities give a value for their molecular weight of (3.4±0.2)×106.
Role of carbohydrate as an antigenic determinant of a glycoprotein from rye-grass (Lolium perenne) pollen
Authors: Howlett B J , Clarke A E
Published: August 2015

The carbohydrate component of Glycoprotein 2 (12% carbohydrate) from rye-grass (Lolium perenne) pollen has saccharide sequences that contribute to its antigenicity. Radioimmunoassay inhibition tests show that the antiserum to this glycoprotein cross-reacts with a number of other plant glycoproteins. In contrast, antiserum to another glycoprotein from rye-grass pollen, Glycoprotein 1 (5% carbohydrate), does not cross-react with any of the test glycoconjugates. Treatment of glycoproteins with sodium metaperiodate (0.02 M, 4 degree C, 6 h, in the dark) causes the loss of their ability to cross-react antigenically with Glycoprotein 2, and a loss of capacity to bind 125I-labelled concanavalin A. The cross-reactivity of this plant glycoprotein with other glycoconjugates imposes limitations on the interpretation of ultrastructural studies aimed at localizing a particular glycoprotein to a cellular site by using fluorescent or ferritin-labelled antisera. A radioimmunoassay inhibition technique for quantitative determination of the amounts of antigens in plants is also described.
Application of jackknife procedures to inter-experiment comparisons of parameter estimates for the Michaelis-Menten equation
Authors: Oppenheimer L , Capizzi T P , Miwa G T
Published: August 2015

The jackknife procedure is introduced as a means of making comparisons among Michaelis-Menten parameter estimates for six different experimental conditions. In addition to providing a solution to the general inter-experimental comparison problem, the jackknife procedure will provide valid parameter estimates even when some of the assumptions usually required for statistical analysis are violated, e.g., the random errors are not normally distributed and the variances are not homogeneous. Other recent variations of the jackknife have also been introduced and briefly investigated: (i) the linear jackknife, which is more efficient computationally, and (ii) the weighted jackknife, which reduces the influence of design points (substrate concentrations) that have an excessive influence on the precision of parameter estimates.
Dehydrogenation by gluconate 6-phosphate dehydrogenase of an isosteric phosphonate substrate analogue
Authors: Roach D J , Harrison R
Published: August 2015

6,7-Dideoxy-D-gluco-heptonic-7-phosphonic acid, the isosteric phosphonate analogue of gluconate 6-phosphate, was prepared by incubation of the corresponding analogue of glucose 6-phosphate with glucose 6-phosphate dehydrogenase and NADP+ in the presence of an enzyme NADPH-NADP+ recycling system. The analogue of gluconate 6-phosphate is a substrate for yeast gluconate 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH 7.5 and 8.0. At both pH values the Km values are approx. 3-fold higher and the Vmax. values approx. 7-fold lower than those of the natural substrate.
Identification, isolation and some properties of lectin from the seeds of Indian coral tree [Erythrina variegata (Linn.) var. orientalis (Linn.) Merrill]
Authors: Datta T K , Basu P S
Published: August 2015

A D-galactose-binding lectin agglutinating human erythrocytes has been purified from the seeds of the Indian coral tree (Erythrina variegata (Linn.) var. orientalis (Linn.) Merrill] by affinity chromatography on acid-treated Sepharose-6B gel. It has a higher reactivity for O-group erythrocytes. The lectin is a glycoprotein having a leucoagglutinating property.
Proteoglycans of bovine periodontal ligament and skin. Occurrence of different hybrid-sulphated galactosaminoglycans in distinct proteoglycans
Authors: Pearson C H , Gibson G J
Published: August 2015

A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.
Fluid-phase interaction of C1 inhibitor (C1 Inh) and the subcomponents C1r and C1s of the first component of complement, C1
Authors: Chesne S , Villiers C L , Arlaud G J , Lacroix M B , Colomb M G
Published: August 2015

Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated.
The reaction of ornithine aminotransferase with ornithine
Authors: Williams J A , Bridge G , Fowler L J , John R A
Published: August 2015

Rat liver ornithine aminotransferase is found to exchange the pro-S hydrogen on the delta-carbon atom of ornithine exclusively, thus showing that the mammalian enzyme transfers the delta-amino group and not the alpha-amino group as has been demonstrated with the plant enzyme [Mestichelli, Gupta & Spenser (1979) J. Biol. Chem. 254, 640-647]. The enzyme also transfers the alpha-amino group of glutamate and the kinetics of the half reactions between the enzyme and both amino acids are compared. Rate and dissociation constants for both reactions are determined.
Co-polymeric glycosaminoglycans in transformed cells. Transformation-dependent changes in the co-polymeric structure of heparan sulphate
Authors: Fransson Lars-Ȧke , Havsmark Birgitta , Chiarugi Vincenzo P.
Published: August 2015

1. Heparan sulphates from normal 3T3 fibroblasts are association-prone as indicated by their affinity for agarose gels substituted with cognate heparan sulphate species. Heparan sulphates from SV40-transformed or polyoma-virus-transformed cells have no affinity for the same gels. 2. Heparan sulphates from the medium, the pericellular and intracellular pools of normal, SV40-transformed and polyoma-transformed 3T3 cells were separated into four subfractions (HS1–HS4) by ion-exchange chromatography. In general, HS1–HS3 were found in cell-derived heparan sulphates, whereas HS3–HS4 were present in the medium. The heparan sulphates from transformed cells were more heterogeneous and of lower charge density than those from the normal counterpart. 3. Degradations via periodate oxidation/alkaline elimination yielded the oligomers glucosamine-(hexuronate–glucosamine)n-R with n=1–5 and a large proportion of N-sulphate groups. There was a large contribution of fragments n=4–5 from heparan sulphates of normal cells. These fragments were less common in low-sulphated heparan sulphates of transformed cells. In the case of medium-drived heparan sulphates all species had a low content of fragments n=4–5. 4. The size distribution of (glucuronate–N-acetylglucosamine)n regions was assessed after deaminative cleavage. It was broad and ranged from n=1–10 for all heparan sulphate species. In the case of medium-derived heparan sulphates there were distinct differences between normal and transformed cells. In the latter chains the N-acetyl-rich segments were both shorter and longer than in the normal case. The shape of the disaccharide peak was consistent with a lower content of O-sulphate in the heparan sulphates from transformed cells. 5. It was concluded that heparan sulphates from medium or transformed cells exhibit the greatest structural deviation from the normal case. The finding of lower proportions of extended, iduronate/glucuronate-bearing, N-sulphate-rich segments in heparan sulphates of transformed cells was particularly interesting in view of the fact that these elements have been associated with ability to self-interact.
Degradation of myofibrillar proteins by trypsin-like serine proteinases.
Authors: Kay J , Siemankowski L M , Siemankowski R F , Greweling J A , Goll D E
Published: August 2015

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.
Isolation and characterization of plasma-membrane glycoproteins from pig epidermis
Authors: King Ian A. , Tabiowo Anne
Published: August 2015

1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000–80000. 2. A large proportion of the marker enzymes, the d-[3H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A–Sepharose 4B and lentil lectin–Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) α-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[3H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie Brilliant Blue-stained components. These were weakly stained by periodic acid/Schiff reagent and were lightly labelled with d-[3H]glucosamine, indicating that they contained less carbohydrate than the four major glycoprotein bands. 5. Chloroform/methanol-extracted plasma membranes and isolated glycoproteins had a similar carbohydrate composition, containing sialic acid, hexosamine, fucose, xylose, mannose, galactose and glucose. Glucose was not enriched in the isolated glycoproteins, suggesting that it may be a contaminant. Xylose, however, was enriched in the isolated glycoproteins. It remains to be established whether this sugar, which is not usually found in plasma-membrane glycoproteins, is a genuine constituent of plasma-membrane glycoproteins in the epidermis.
Alkyl selenosulphates (seleno Bunte salts). A new class of thiol-blocking reagents.
Authors: Scarf A R , Cole E R , Southwell-Keely P T
Published: August 2015

Potassium benzyl selenosulphate and potassium p-nitrobenzyl selenosulphate were shown to be powerful inhibitors of the thiol-dependent enzymes glutathione reductase and papain, but to have no effect on the serine-dependent proteinase trypsin. By contrast, potassium benzyl thiosulphate and potassium p-nitrobenzyl thiosulphate, at much higher concentrations, have virtually no effect on any of the enzymes. The selenosulphates show characteristics of both reversible non-competitive and irreversible inhibition. On the basis of model reactions in which the selenosulphates react instantly with cysteine, it is suggested that they form labile selenosulphide derivatives with the enzymes, but that these derivatives may be broken down either by the normal functioning of the enzyme (in the case of glutathione reductase) or by the approaching substrate (in the case of papain). Continued inhibition of the enzymes requires a stoicheiometric excess of inhibitor over enzyme.
Three functionally different cytochrome b redox centres in pigeon heart mitochondria.
Authors: Rasmussen U F , Rasmussen H N , Jørgensen B M
Published: August 2015

Three functionally different cytochrome b redox centres, apparently of high metabolic activity, were detected in intact pigeon heart mitochondria; cytochrome b(1), b(m) and b(h), with maxima of absorption at 556.6 (State 5), 560.6, and 564.5 nm, respectively (alpha-bands, 77K). 2. Cytochrome (b(l) was reduced in the presence of either antimycin or HQNO (2-heptyl-4-hydroxyquinoline N-oxide). The absorption maximum was shifted by dithionite, cyanide, NNN‘N’-tetramethyl-p-phenylenediamine + ascorbate, HQNO and antimycin. The spectra obtained on simultaneous or successive addition of HQNO and antimycin favoured the assumption of a common binding site for the two inhibitors. 3. Cytochrome b(m) was reduced in the presence of HQNO, but not in the presence of antimycin. No shifts of absorption maximum was observed. 4. Cytochrome b(h) was reduced in the presence of antimycin. HQNO was unable to cause reduction of this cytochrome by endogenous substrates. The absorption maximum was shifted to lower wavelength by organic solvents. It was inseparable from that of cytochrome b(m) in the presence of 0.4% ethanol. 5. The pattern of reduction in the presence of HQNO or antimycin demonstrates the functional difference of the three redox centres and appears incompatible wih a linear respiratory chain.
The identification of the weak oestrogen equol [7-hydroxy-3-(4′-hydroxyphenyl)chroman] in human urine
Authors: Axelson M. , Kirk D. N. , Farrant R. D. , Cooley G. , Lawson A. M. , Setchell K. D. R.
Published: August 2015

The identification (by gas chromatography–mass spectrometry and n.m.r.) for the first time of the weak oestrogen equol [7-hydroxy-3-(4′-hydroxyphenyl)chroman] in human urine is described. Preliminary results of its quantitative excretion in urine are reported and the potential significance of the occurrence of this compound is discussed.
Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells
Authors: Sasak Wlodzimierz , Herscovics Annette , Quaroni Andrea
Published: August 2015

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-3H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [3H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.
Action of rat liver cathepsin L on collagen and other substrates.
Authors: Kirschke H , Kembhavi A A , Bohley P , Barrett A J
Published: August 2015

1. It has been found that cathepsin L is very susceptible to loss of activity through autolysis. When this is prevented by purification and storage of the enzyme as its mercury derivative, preparations are obtained with higher specific activity than previously. 2. Active-site titration shows, however, that even the new purification method does not give preparations in which the enzyme is 100% active. 3. Benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide has been discovered to be a very sensitive substrate for cathepsin L. Like all other known substrates for cathepsin L, however, it is also cleaved by cathepsin B. 4. Cathepsin L degrades insoluble collagen at pH 3.5 over 5-fold faster than at pH 6.0. The specific activity at pH 3.5 is 5-10-fold higher than that of cathepsin B (rat or human) or bovine spleen cathepsin N (‘collagenolytic cathepsin’). 5. Qualitatively, the action of cathepsin L on collagen is similar to that of cathepsins B and N, i.e. selective cleavage of terminal peptides leads to conversion of beta- and higher components mainly to alpha-chains.
Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products.
Authors: Bergh M L E , Koppen P L , Van den Eijnden D H
Published: August 2015

High-pressure liquid chromatography was used to identify the sialo-oligosaccharide products obtained after sialylation of [14C]Gal-GalNAc-protein in vitro by an ovine submaxillary-gland microsomal fraction. Among other products, two isomeric trisaccharides could be identified. NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol and Gal beta 1 leads to 3-(NeuAc alpha 2 leads to 6)GalNAcol respectively, indicating that ovine submaxillary gland contains two sialyltransferases acting on mucin-type acceptors, a beta-galactoside alpha 2 leads to 3 sialyltransferase and a N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. This conclusion was fully supported by methylation analysis of the two trisaccharide products.
The molecular composition of the volutin granule of yeast
Authors: Jacobson L , Halmann M , Yariv J
Published: August 2015

The volutin granule was isolated from yeast by disruption of freeze-dried cells in an organic solvent and density-gradient-gradient centrifugation. The granule is composed of two types of macromolecule, a linear-chain polyphosphate and four basic proteins, of molecular weights ranging from 10 000 to 20 000. In the dissolved granule these macromolecules are in a complex that is uniform by hydrodynamic criteria (s20,w = 22.3 S). The polyphosphate separated from this complex gives a single 31P n.m.r. resonance and in the analytical ultracentrifuge behaves as a monodisperse solute of molecular weight 245 000 +/- 1000. In the 31P n.m.r. spectrum of yeast used for its isolation, this polyphosphate accounts for 14% of total cell polyphosphate.
A new method for the extraction and purification of K99 pili from enterotoxigenic Escherichia coli and their characterization
Authors: Altmann K , Pyliotis N A , Mukkur T K S
Published: August 2015

It was found that K99 pili from enterotoxigenic Escherichia coli (of bovine origin) could be extracted by treatment with 3M-KSCN solution. The K99 pili were purified by preparative isoelectric focusing to apparent homogeneity as judged by the presence of a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the molecular weight of this component was calculated to be 12 600 +/- 300. This indicated that the K99 pili were composed of a single subunit. On analytical ultracentrifugation, a single boundary with an s20,w of 12.2 S at a concentration of 0.42 mg/ml was observed. The average length of purified pili at zero concentration was approx. 160 nm and the diameter was 7.4 +/- 0.6 nm. Amino acid analysis of the purified K99 pili revealed that sulphur-containing amino acids, cysteine and methionine, were absent. Aromatic amino acids, phenylalanine and tyrosine, previously reported to be absent [Isaacson (1977) Infect. Immun. 15. 272-279], constituted 7.14% of the total amino acid residues present. On immunoelectrophoresis, purified K99 pili migrated towards the cathode and caused mannose-resistant haemagglutination of horse, but not of sheep or guinea-pig, red blood cells. Pili from enterotoxigenic E. coli of porcine and human origin and from another bacterial species, namely Fusiformis nodosus, could also be extracted by the treatment of respective micro-organisms with 3 M-KSCN.
Physicochemical properties of avian tracheal mucus
Authors: Mian N , Anderson C E , Pope A J , Kent P W
Published: August 2015

Dual-radiolabelled avian tracheal secretions were obtained by giving Na235SO4 and D-[1-3H]glucosamine simultaneously into the lumen of the trachea in preparations in vitro. These secretions comprised fibrillar, gelatinous and soluble-phase mucins. These were eluted as single components in the non-retarded fractions from Bio-Gel A-15m. Although no evidence of the presence of subunit structure was found, chemical and radiolabelling analyses showed a high degree of internal inhomogeneity among the three types of mucins. The differences among these mucins could be attributed to the chemical nature of their constituent glycoproteins. Glycoprotein fractions separated by ion-exchange chromatography were found to contain sulphate and N-acetylneuraminic acid residues in differing amounts. The overall acidic properties appeared to be correlated with ester sulphate content. A close similarity in the carbohydrate composition and a reciprocal relationship between the total ester sulphate residue contents and 35S- and 3H-labelling suggested that, in addition to stepwise glycosylation and sulphation, some pre-existing sulphated oligosaccharides might have been utilized for the synthesis of acidic glycoproteins.
The binding of human liver acid β-galactosidase to wheat-germ lectin is influenced by aggregation state of the enzyme
Authors: Heyworth C M , Wynn C H
Published: August 2015

At low ionic strength and pH 6.0 human liver acid beta-galactosidase exists in two aggregation states, monomeric and multimeric. These species can be separated on wheat-germ lectin-Sepharose, Cellogel electrophoresis and gel filtration on Sephadex G-200, and are not normally interconvertible. On re-application of either form to wheat-germ lectin-Sepharose the equilibrium is re-established and the two forms are interconverted.
Enzymic coupling of acylhydrolase and prostaglandin synthase activities in subcellular fractions from rabbit renal medulla
Authors: Erman Arie , Azuri Ruth , Raz Amiram
Published: August 2015

We have recently shown that mitochondrial and plasma-membrane fractions from kidney medulla possess Ca2+-stimulated acylhydrolase and prostaglandin synthase activities. The nature of the enzymic coupling between the Ca2+-stimulated arachidonic acid release and its subsequent conversion into prostaglandins was investigated in subcellular fractions from rabbit kidney medulla. Plasma-membrane, mitochondrial and microsomal fractions were found to have similar apparent Km values for conversion of added exogenous arachidonate into prostaglandins. The rate of prostaglandin biosynthesis (Vmax.) from added arachidonic acid in the microsomal fraction was approx. 2-fold higher than in the other subcellular fractions. In contrast, prostaglandin E2 synthesis from endogenous arachidonate in plasma-membrane and mitochondrial fractions was 3–4-fold higher than in microsomes. Furthermore, Ca2+stimulated endogenous arachidonate deacylation and prostaglandin E2 generation in the former two fractions but not in microsomes. In mitochondrial or crude plasma-membrane fractions, in which prostaglandin biosynthesis was inhibited with aspirin, arachidonate released from these fractions was converted into prostaglandins by the microsomal prostaglandin synthase. Thus an intracellular prostaglandin generation process that involves inter-fraction transfer of arachidonic acid can operate. Prostaglandin generation by such an inter-fraction process is, however, less efficient than by an intra-fraction process, where arachidonic acid released by mitochondria or crude plasma membranes is converted into prostaglandins by prostaglandin synthase present in the same fraction. This demonstrates the presence of a tight intra-fraction enzymic coupling between Ca2+-stimulated acylhydrolase and prostaglandin synthase enzyme systems in both mitochondrial and plasma-membrane fractions.
The production and properties of an antiserum to potato (Solanum tuberosum) lectin
Authors: Ashford D , Allen A K , Neuberger A
Published: August 2015

Precipitation of potato (Solanum tuberosum) lectin by antisera was not affected by treatments that abolish lectin activity. An antiserum precipitated glycosylated derivatives of the lectin but not a deglycosylated peptide. The haemagglutination inhibition titre of this antiserum was not affected by removing anti-glycopeptide antibodies. This evidence suggests that the antiserum contains two populations of antibodies, specific for different domains of the lectin.
Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes
Authors: Burchell B
Published: August 2015

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.
Use of the F test for determining the degree of enzyme-kinetic and ligand-binding data. A Monte Carlo simulation study
Authors: Burguillo F J , Wright A J , Bardsley W G
Published: August 2015

1. Initial-rate data were simulated for 13 representative enzyme mechanisms with the use of several distributions of rate constants in order to locate conditions leading to v([S]) curves in physiological ranges of substrate concentration. 2. In all, 420 sets of such v([S]) curves were generated with the use of several choices of substrate concentration range (two, three or four orders of magnitude), number of experimental points (10, 15 or 20), error on v (5-10%) and standard deviation on v (5-9%) in order to simulate experimental results in a number of possible ways. 3. Curve-fitting was carried out to rational functions of degree 1:1, 2:2, …, 5:5 until there was no statistically significant decrease in the sum of weighted squared residuals as judged by the F test at 95% and 99% confidence levels. 4. It was checked whether the non-linear regression program had located a good minimum in the sum of squares by also fitting the data with the correct values of parameters as starting estimates. 5. A similar procedure was adopted with 110 sets of binding data simulated for 11 models, and the F test was used to see if fractional-saturation data generated by a binding polynomial of order n could be adequately fitted by one of order m, m less than n. 6. From the 530 simulations the F test was successful in fixing the correct degree with a probability of 0.62 at the 95% confidence level, but this fell with increase in degree as follows: 1:1 (0.98), 2:2 (0.71), 3:3 (0.43) and 4:4 (0.34), the first numbers being the degree of the rate equation and those in parentheses referring to the 95% confidence level. 7. It made little difference whether the 95% or the 99% confidence level was consulted, as there were very few borderline cases. 8. The chance of detecting deviations from Michaelis-Menten kinetics, i.e. terms of at least second-order in a rate equation of degree n:n, n greater than 1, was estimated to be about 0.8. 9. The probability of the F test leading to a spurious result due to error in the data was found to be about 0.04. 10. The probability with which 4:4 mechanisms can lead to v([S]) plots with no, one, two or three turning points was computed, and it was established that there is a small but finite chance that the increase in degree that occurs in some mechanisms when ES in equilibrium EP interconversions are explicitly allowed for can be detected by the F test.
The role of glandular kallikrein in the formation of a salivary proline-rich protein A by cleavage of a single bond in salivary protein C.
Authors: Wong R S C , Madapallimattam G , Bennick A
Published: August 2015

An enzyme was purified from human parotid saliva that can cleave a single arginine-glycine peptide bond between residues 106 and 107 in human salivary proline-rich protein C, hereby giving rise to another proline-rich protein A, which is also found in saliva. The enzyme was purified 2400-fold. It cleaved salivary protein C at the rate of 59 micrograms of protein/h per microgram of enzyme and had amino acid composition, molecular weight and inhibition characteristics similar to those reported for human salivary kallikrein. Confirmation that the enzyme was kallikrein was demonstrated by its kinin-generating ability. Histochemical evidence indicates that a post-synthetic cleavage of protein C by kallikrein would have to take place during passage of saliva through the secretory ducts. In secreted saliva, cleavage of salivary protein C can only be observed after 72 h incubation. In addition, there is no effect of salivary flow rate on the relative amounts of proteins A and C in saliva. On the basis of the experimental observations, it is proposed that in vivo it is unlikely that kallikrein secreted from ductal cells plays a significant role in converting protein C into protein A.