Carbohydrates in protein. 4. The determination of mannose in hen's-egg albumin by radioisotope dilution

François, Camille; Marshall, Ruth; Neuberger, A.

doi:10.1042/bj0830335

Cited by 336 publications

(62 citation statements)

References 28 publications

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“…Glucose and galactose bound to hydroxylysine were determined after hydrolysis with 2 M KOH. The orcinol reaction was used for the colorimetric determination of the hexose content [21] and a mixture of glucose and galactose (1 : 1) served as the standard. Thin-layer chromatography of hexoses on silica gel 60 plates (solvent system benzene/butan-I-ol/acetone/water, 2: 3 : 4: 1, v/v) was carricd out with hydrolysed samples (4 M HCI, 3 h, 1 10 ' C ) and individual sugars were detected by reaction with diphenylamineaniline [22,23].…”

Section: Methodsmentioning

confidence: 99%

7‐S Collagen: Characterization of an Unusual Basement Membrane Structure

Risteli

Timpl

Bächinger

et al. 1980

European Journal of Biochemistry

294

126

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A new type of collageneous structure, tentatively named 7-S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistence towards further degradation by bacterial collagenase at 20°C. 7-S collagen showed an amino acid composition typical of basement membrane collagen and contained 22 carbohydrate mainly as glucosyl-galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with t, = 48 '-C and 70 "C. The more stable domain could be isolated as an homogeneous fragment ( M , = 225000) after a second digestion with collagenase at 37 "C. This fragment contained all the disulfide bonds (42 Cys/lOOO residues) of the original molecule. Electron microscopy showed a rod-like structure in agreement with the hydrodynamic properties of 7-S collagen. The dimensions of these peptides were 3 x 95 nm (long form) and 3.4 x 40-50 nm (short form).Complete reduction of 7-S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27 000 -153 000 which differ from each other by M , i ncrements 25 000 -27 000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross-linked or represent fragments produced during proteolytic treatments. Complete reduction of 7-S collagen under non-denaturing conditions lowered the thermal transition of the triple helix to 48 'C but did not change its molecular weight except when exposed to dissociating solvents. 7-S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37 'C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross-reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7-S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.Basement membranes are extracellular matrices widely distributed in the body. They appear ultrastructurally amorphous and consist of a complex mixture of non-collageneous and collageneous glycoproteins. The major collageneous component was originally named type IV collagen [l] and was found to ~ Ahbveviutions. CD, circular dichroism; I,, ...

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Section: Methodsmentioning

confidence: 99%

7‐S Collagen: Characterization of an Unusual Basement Membrane Structure

Risteli

Timpl

Bächinger

et al. 1980

European Journal of Biochemistry

294

126

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“…Aliquots of density fractions collected after ultracentrifugation (density from 1.29 to 1.59 g·mL -1 ) were evaporated (Speed-Vac SC 110, Savant Instruments, New York, USA), dissolved in Tris buffer (10 mM, pH 7.5) containing 35 mM sodium dodecyl sulfate (SDS) and analyzed for total protein content using the Lowry method with serum albumin as a standard [13] and for total glycoprotein using the orcinol-sulfuric acid procedure, with galactose as the standard [14]. Nucleic acids were monitored at an optical density of 260 nm (U Vikon 922, Kontron Instruments, UVK-Lab, Trappes, France).…”

Section: Analysis Of Density Fractions and Purified Glycoproteinmentioning

confidence: 99%

Estimation of ileal output of gastro-intestinal glycoprotein in weaned piglets using three different methods

et al. 2004

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-Mucin is the main constituent of gastrointestinal mucus and is responsible for its physicochemical and physiological properties. Previous studies have suggested that this glycoprotein represents a major component of undigested endogenous protein at the ileum. The aim of the study was to estimate the ileal output of this glycoprotein using three methods: direct ELISA, hexosaminebased method and ethanol precipitation. For setting up the ELISA assay, the glycoprotein was isolated from intestinal mucus scraping by cesium chloride density gradient ultracentrifugation and a rabbit hyperimmune plasma was raised against the purified glycoprotein. Ileal outputs of hexosamine and glycoprotein were measured in weaned piglets fed a control diet (C) based on casein or diets which contained 50% crude protein supplied by white (WCP) or black (BCP) chickpea. The hexosamine output was higher (P < 0.05) with the WCP diet (2.3 and 1.5 g·kg -1 of dry matter intake for glucosamine and galactosamine, respectively) than with diet C (1.1 and 0.7 g·kg -1 of DMI). The hexosamine-based and ethanol precipitation methods, but not the ELISA, showed significant differences between the diet treatments (P < 0.05). Although hexosamine-based and ethanol precipitation methods for the estimation of ileal glycoprotein appeared to be more satisfactory than the developed ELISA to display diet effects in this study, it remains to be determined whether the higher glycoprotein data variability observed with ELISA reflects the actual biological variability of the phenomenon or not.small intestine / methods / glycoprotein / piglet

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“…This is a potential source of error in the analysis of milk, such as that of marsupials, which contains unequal amounts of galactose and glucose. One is faced with the same problem with at least three other methods for total hexose; With the orcinol-sulfuric aCid method, galactose gives a higher colour yield than glucose (Francois et al 1962), whereas with both the anthrone and the cysteine-sulfuric acid methods, it gives a lower colour yield (Morris 1948;Dische 1962). Fig.…”

Section: Determination Of Milk Carbohydratementioning

confidence: 99%

Milk Carbohydrates of Marsupials II. Quantitative and Qualitative Changes in MiIk Carbohydrates During Lactation in the Tammar Wallaby (Macropus Eugenii)

Messer¹,

Green²

1979

Aust. Jnl. Of Bio. Sci.

125

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Milk was collected at various stages of lactation from a group of tammar wallabies, M. eugenii, in which parturition had been synchronized.The milk carbohydrate was determined by a phenol-sulfuric acid method which had been modified to give equal colour yields for galactose and glucose. The mean carbohydrate content increased gradually during the first 6 months of lactation to a peak of 13 g hexose/100 ml of milk, but then fell rapidly to much lower values, over the following 2 months.Throughout lactation, galactose was the predominant monosaccharide constituent of acid hydrolysates of the milk carbohydrate. Glucose, glucosamine, galactosamine and sialic acid were th,~ only other monosaccharides present.Qualitative changes were investigated by gel filtration and thin-layer chromatography. During the first 6 months post partum the milk carbohydrate was composed of a variety of oligosaccharides induding lactose, but from 8 months onwards it consisted mainly of free monosaccharides. Between 6 and 8 months an intermediate pattern was observed, i.e. a mixture of lower oligosaccharides and fre:e monosaccharides. In two animals which suckled both a new-born pouch young and a young at foot, the mammary gland supplying the new-born secreted milk which was rich in oligosaccharides, whereas that supplying the young at foot produced milk in which the carbohydrates were mainly free monosaccharides, and which had a much lower carbohydrate content.

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Carbohydrates in protein. 4. The determination of mannose in hen's-egg albumin by radioisotope dilution

Cited by 336 publications

References 28 publications

7‐S Collagen: Characterization of an Unusual Basement Membrane Structure

7‐S Collagen: Characterization of an Unusual Basement Membrane Structure

Estimation of ileal output of gastro-intestinal glycoprotein in weaned piglets using three different methods

Milk Carbohydrates of Marsupials II. Quantitative and Qualitative Changes in MiIk Carbohydrates During Lactation in the Tammar Wallaby (Macropus Eugenii)

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