IgE-reactive 60 kDa glycoprotein occurring in wheat flour.

A new IgE-reactive glycoprotein with a molecular size of 60 kDa was isolated from wheat flour. The N-terminal amino acid sequence of the protein was LDPDESEXVTRYFRIR. The 8th amino acid residue would have been Asn to which the peroxidase-type glycochain was attached. The IgE-binding activity of the glycoprotein was rendered negligible by the enzymatic treatment applied for hypoallergenic flour production.     

Studies on Amino Acid Contents of Processed Soybean

Effect of heating on the nutritive value of defatted soybean flour has been investigated by animal experiments. Loss due to heat degradation was evaluated in two ways. In the first method, the amino acids lost during overheating were supplemented by cystine and mixture of lysine, arginine, tryptophan, and serine at dietary levels of 1.6% nitrogen, and cystine and mixture of those amino acids plus histidine at dietary levels of 3.2% nitrogen. The other procedure adopted was the absorbent test used with amino acid mixtures based on the pattern of amino acids released by pancreatic hydrolysis of unheated, properly heated, and overheated defatted soybean flour at 6 and 120 hr hydrolysis.

At 1.6% dietary nitrogen level, the nutritive value of overheated soybean flour increased by supplementation with cystine and amino acid mixture, but at the 3.2% nitrogen level only cystine was effective. Supplementation of lost amino acids to overheated flour did not restore the nutritive value to that of the properly heated flour. Based on the amino acids released by pancreatic hydrolysis of unheated, properly heated, and overheated soybean flour after 6 and 120 hr reaction, amino acid mixtures were prepared and tested for their nutritive value. While the nutritive value of amino acid mixture prepared based on the pattern of amino acid liberated by 6 hr digestion of unheated, properly heated, and overheated flour did not show similar trend to that of 3 kinds of flour itself, the nutritive value of the amino acid mixture prepared after the data obtained by 120 hr digestion agreed well with the trend of unheated or heated soybean flour.

The nutritive value was also measured by the nitrogen balance of test animals.     

Consumption of hypoallergenic flour prevents gluten-induced airway inflammation in Brown Norway rats

Brown Norway rats were immunized with gluten, and then fed a diet containing hypoallergenic flour or an amino acid mixture. The rats were then made to inhale a solubilized gluten to induce gluten-specific bronchial asthma. The antibody levels in the serum of rats were measured by ELISA, and cell counts were done on cytospin preparations of bronchoalveolar lavage fluid. Body weight was decreased after allergen challenge in rats fed the amino acid mixture but not in rats fed the hypoallergenic flour. Antibody levels in the serum were significantly lower in rats fed hypoallergenic flour than in those fed the amino acid mixture. Differential cell counts in the bronchoalveolar lavage fluid showed that the numbers of eosinophils, lymphocytes, and neutrophils were significantly lower in rats fed the hypoallergenic flour than in those fed the amino acid mixture. These results suggest that hypoallergenic flour actively suppresses the allergic reactions, probably by inducing oral tolerance.     

Tamm–Horsfall urinary glycoprotein. The chemical composition

1. A revised amino acid and carbohydrate composition of human Tamm-Horsfall glycoprotein is presented. 2. No significant differences were obtained in the amino acid composition of Tamm-Horsfall glycoprotein isolated from patients with cystic fibrosis. 3. The glycoprotein was shown to possess a high half-cystine content of 1 per 11-12 amino acid residues, which has been confirmed by performic acid oxidation and S-alkylation with iodoacetate and iodoacetamide. No thiol groups were detected in the glycoprotein. 4. Treatment of the glycoprotein with 0.5m-sodium hydroxide at 4 degrees C for 2 days did not release heterosaccharide material, which suggests that the predominant carbohydrate-protein linkages present are not of the O-glycosidic type. 5. No N-terminal amino acid was detected in the glycoprotein.     

Preparation and characterization of armadillo submandibular glycoproteins.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.     

Isolation and characterization of human platelet glycoprotein IX

Human platelet glycoprotein IX is a small (Mr 17,000) surface glycoprotein present in the plasma membrane as a 1:1 non-covalent complex with glycoprotein Ib (Mr 165,000). Glycoprotein IX was purified to near homogeneity by sequential wheat germ agglutinin and immuno-affinity chromatography, followed by gel filtration chromatography under denaturing conditions. Purified glycoprotein IX was characterized by determination of its amino acid composition and its NH2-terminal amino acid sequence (thr-lys-asp-xxx-pro-ser-xxx-leu-thr-(thr)2-arg-ala-(leu)-(glu)-xxx-met- gly), and monospecific anti-glycoprotein IX antibody was prepared. The study outlines a useful approach for separating and characterizing glycoprotein IX, free from other members of the glycoprotein Ib complex.     

Further Studies of Pollen Wall Glycoproteins in Cucurbita pepo L.

Samples of pollen wall protein of Cucurbita pepo were prepared as reported in previous paper. Gas chromoatographic analyses snowed that the carbohydrate fraction of the pollen wall glycoprotcin contained 20.4% rhamnose, 15.3% fucose, 11% mannose, 11% galactose, 31% glucose, 4% arabinose and traces of xylose. The glycoproteins were further purified by Con. A affinity chromatography, Isoelectric focussing electrophoresis of the purified sample showed 3 PAS-positive bands, with respective PI 5.2, 6.0 and 6.3. The glycoprotein samples were subjected to hydrolysis with 6N HC1. After hydrolysis, the mixture was analyzed for amino acid composition with Backman 121-MB automatic amino acid analyzer, Results show the amino acid composition of the 3 glycoprotein was very similar, They all have glycine, glutamic acid and serine as their major component (these three amino acids constitute 50–60% of the total amino acids); and they all contain very small amount of methionine, phenylalanine, isoleucine and tyrosine. The lysine content of each glycoprotein is consistent with its respective PI, the glycoprotein which contains more lysine has higher PI.     

Chemical and Nutritional Properties of Hypoallergenic Wheat Flour

The chemical and nutritional properties were investigated of hypoallergenic wheat flour (HWF) prepared by the cellulase-actinase treatment. HWF was composed mainly of oligopeptides and free amino acids, and its average molecular weight was lower than 1,000. Feeding tests on rats showed that, with respect to the PER, GOT and GPT activities and other nutritional indices, the HWF diet was almost equivalent to the control diet which had been prepared from normal wheat flour (NWF). No abnormality was apparent in the main organs after the HWF diet had been fed for 3 weeks. The small intestinal absorption of the HWF diet was found normal by measuring the free amino acid concentration in the intestinal tract and in the portal vein plasma. These data suggest that te absorption of amino acids from the HWF diet was comparable with or more efficient than that from a simulated free amino acid diet.     

Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a computer program which determined optimal alignment. An amino acid identity of approximately 20% was found between infectious hematopoietic necrosis virus and the two vesicular stomatitis virus serotypes and between infectious hematopoietic necrosis virus and rabies virus. The positions and sizes of the signal sequence and transmembrane domain and the possible glycosylation sites were determined.     

Chemical and nutritional properties of hypoallergenic wheat flour

The chemical and nutritional properties were investigated of hypoallergenic wheat flour (HWF) prepared by the cellulase-actinase treatment. HWF was composed mainly of oligopeptides and free amino acids, and its average molecular weight was lower than 1,000. Feeding tests on rats showed that, with respect to the PER, GOT and GPT activities and other nutritional indices, the HWF diet was almost equivalent to the control diet which had been prepared from normal wheat flour (NWF). No abnormality was apparent in the main organs after the HWF diet had been fed for 3 weeks. The small intestinal absorption of the HWF diet was found normal by measuring the free amino acid concentration in the intestinal tract and in the portal vein plasma. These data suggest that the absorption of amino acids from the HWF diet was comparable with or more efficient than that from a simulated free amino acid diet.     

Amino acid sequence and location of the disulfide bonds in bovine beta 2 glycoprotein I: the presence of five Sushi domains.

beta 2 glycoprotein I is a plasma protein with the ability to bind with various kinds of negatively charged substances. The complete amino acid sequence and the location of all the disulfide bonds of bovine beta 2 glycoprotein I were determined. Bovine beta 2 glycoprotein I consists of 326 amino acid residues with five asparagine-linked carbohydrate chains. Homology with the human protein was calculated to be 83%. Eleven disulfide bonds in bovine beta 2 glycoprotein I constitute four characteristic domains, Sushi domains, and one modified form of a Sushi domain.     

Purification and some chemical properties of 30 kDa Ginkgo biloba glycoprotein, which reacts with antiserum against beta 1-->2 xylose-containing N-glycans

From the seeds of Ginkgo biloba, a glycoprotein, which is a major component that reacts with an antiserum against beta 1-->2 xylose-containing N-glycans, has been purified and characterized. The N-terminal amino acid sequence of the purified glycoprotein was H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-L-L-F-G-Q-. The molecular mass was estimated to be 17 kDa and 16 kDa by SDS-PAGE under reducing conditions, however, the molecular mass of this glycoprotein in the native state was 30,762 by MALDI-TOF MS, suggesting that this glycoprotein consists of two subunits; one is glycosylated and the other is not. The structure of N-glycan linked to this glycoprotein (designated 30 kDa GBGP) was identified as Man3Fuc1Xyl1GlcNAc2, which is the predominant N-glycan linked to the storage glycoproteins in the same seeds (Kimura, Y et al. (1998) Biosci. Biotechnol. Biochem. 62, 253-261). From the peptic digest of the carboxymethylated glycosylated subunit, one glycopeptide was purified by RP-HPLC and the amino acid sequence was identified as H-K-A-N-N(Man3Fuc1Xyl1Glc-NAc2)-V-T-V-A-F, which corresponded to the N-terminal amino acid sequence.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

Proteolytic activity of sourdough bacteria

Summary Proteolytic activity during the fermentation of sourdough results in an increase in amino acid content. The proteolysis is caused by flour enzymes, microbial enzymes of flour and by sourdough bacteria. The results indicate that the lactic acid bacteria of sourdough are important for proteolytic activity during the fermentation of sourdough. This proteolytic activity depends on the species of bacteria. Homo- and heterofermentative sourdough bacteria effect different amino acid spectra. Qualitative and quantitative differences in sulphur-containing, cyclic and hydroxy amino acids have been observed. The proteolytic process can be influenced by fermentation conditions, especially the temperature. A lesser effect is observed in the dough yield (flour-water relationship). From experiments with different strains and species of lactic acid bacteria, it is concluded that only one third of the proteolytic activity in sourdough is based on proteases from the flour.Publication-No. 5592 of Federal Research Centre for Cereal and Potato Processing, Detmold, Federal Republic of GermanyPaper presented at the Third International Conference Rye and Triticale, Poznan, Poland, 13–14 May 1987     

Characterization of human angiotensinogen

In this study of the physical and chemical properties of human angiotensinogen were determined. Human angiotensinogen is a glycoprotein containing 14% carbohydrate. The molecular weight as determined by sedimentation equilibrium studies was 56,800. A higher molecular weight was obtained on sodium dodecyl sulfate electrophoresis. Ferguson-type plots indicated that angiotensinogen is another glycoprotein which behaves anomalously on sodium dodecyl sulfate electrophoresis. The COOH-terminal amino acid was found to be serine while two NH2-terminal amino acids, alanine and aspartic acid (or asparagine), were detected. The specific angiotensin I content of angiotensinogen preparations can vary considerably with no effect on the apparent homogeneity of the isolated protein. A protein with negligible angiotensin I content has been obtained from a preparation of human angiotensinogen. The COOH-terminal amino acid of this protein was serine while the only NH2-terminal amino acid detected was alanine.     

Association of vesicular stomatitis virus glycoprotein with virion membrane: characterization of the lipophilic tail fragment.

The proteolytic enzyme, thermolysin, degraded the external segment of the membrane glycoprotein of intact vesicular stomatitis (VS) virions but left behind a small nonglycosylated fragment, presumably embedded in the virion membrane. Other proteases generated membrane-associated glycoprotein fragments differing somewhat in molecular weight. The thermolysin-resistant, virion-associated fragment, which can be selectively solubilized by either Triton X-100 or chloroform/methanol, has a molecular weight of 5,200. Amino acid analysis of the glycoprotein fragment reveals a preponderance of hydrophobic amino acids (64% of the residues); the amino-terminal amino acid is alanine as determined by dansylation. Cyanogen bromide digestion of the tail fragment generated two peptides, confirming the presence of one methionine residue per thermolysin-resistant glycoprotein fragment. The secondary structure of this glycoprotein tail peptide is maintained by at least one disulfide bridge. Thermolysin treatment is isolated VS viral glycoprotein in the presence of Triton X-100 also generated a hydrophobic peptide fragment which is very similar to the virion-associated glycoprotein fragment. The amino acid terminus of intact glycoprotein was also found to be alanine as was its dansylated Triton-micellar fragment that resisted thermolytic degradation; this finding suggests that the amino-terminal end of the VS viral glycoprotein is embedded in the virion membrane. These results suggest that the VS viral glycoprotein is an amphipathic molecule, the hydrophilic portion of which contains all the carbohydrate and a lipophilic tail segment which forms lipid or detergent micelles, thus rendering it resistant to proteolysis.     

Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

1. Tamm-Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm-Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000+/-6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000+/-6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm-Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm-Horsfall antiserum.     

Changes in Microsporum gypseum Mycelial Wall and Spore Coat Glycoproteins During Sporulation and Spore Germination

Ethylenediamine-soluble glycoproteins were extracted from isolated Microsporum gypseum hyphal walls during sporulation and from spore coats before and after germination. This study was carried out to identify a sporulation-specific cell wall protein that possibly served as a substrate for the alkaline protease which initiated the macroconidial germination of this fungus. Analyses revealed that water-insoluble glycoprotein accounted for 10% of the ungerminated spore coat but only for 4 to 5% of the mycelial wall dry weight. This fraction was modified in its amino acid composition during sporulation, and it decreased in protein content during spore germination. Water-soluble glycoprotein, which accounted for approximately 3 to 3.5% of either the spore coat or mycelial wall dry weight, was of similar amino acid composition from both sources and did not decrease in protein content upon spore germination. The water-insoluble glycoprotein was found to be rich in leucine, aspartic acid, glycine, glutamic acid, and phenylalanine residues. The water-soluble glycoprotein was rich in proline, threonine, glycine, serine, glutamic acid, and alanine.     

Antigenic structure of rabies virus glycoprotein: ordering and immunological characterization of the large CNBr cleavage fragments.

After rabies virus glycoprotein was treated with CNBr, the peptide mixture was fractionated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CNBr-cleaved peptide fragments were resolved into seven peptide bands under reducing conditions and six peptide bands under nonreducing conditions. The isolated nonreduced polypeptides were further analyzed by electrophoresis under reducing conditions. The N-terminal amino acid sequences were determined for the peptides in each of the isolated bands. The sequence data identified eight CNBr peptides and allowed the peptide fragments to be ordered within the deduced amino acid sequence of the glycoprotein. Analysis of the nonreduced CNBr peptides revealed two conformations of the glycoprotein. Two CNBr peptide fragments were specifically immunoprecipitated with a hyperimmune anti-rabies glycoprotein serum. These two and one other CNBr peptide induced the production of rabies virus-neutralizing antibodies, indicating the existence of at least three distinct antigenic sites on the rabies virus glycoprotein.     

Studies on Stigma Pellicle Glycoproteins of Raphanus sativus L.

Stigma surface diffusates of Raphanus sativus were subjected to polyacrylamide gel electrophoresis. There appeared protein bands, one of which gave positive PAS reaction, indicating it was glycoprotein. SDS polyacrylamide gel electrophoresis showed that the stigma diffusates contained many protein bands. By comparing their mobilities with those of standard proteins of known molecular weights, the molecular weights of some of the major fractions were estimated to be 15000, 30000—46000 and 70000 daltons. After Schiff reagent staining two main glycoprotein fractions appeared on the SDS gel electrophoretic pattern. Their molecular weights were estimated to be lower than 15000 and higher than 100000 daltons. By using acrylamide gel isoelectric focusing method, it was found that the stigma surface diffusates contained an acidic glycoprotein with pH of about 3.7. The amino acid composition of the purified stigma glycoprotein was determined with amino acid analyzer. Glycine, glutamic acid, serine, aspartic acid were some of the predominant amino acids. The diffusates were analysed by gasliquid chromatography for sugars. Results showed that the carbohydrate fraction of the glycoprotein consisted of arabinose 17.3%; galactose 19.1%, xylose 8.1%, mannose 5.4%, glucose 23.7%, rhamnose and/or fucose 26.4%. In the stigma surface diffusates of Raphanus sativus, the content of protein was estimated to be 16% and that carbohydrate was 11%.