In vitro mucosal digestion of synthetic gliadin-derived peptides in celiac disease

Two celiac-active synthetic peptides derived from the A-gliadin structure corresponding to residues 8–19 (LQPQNPSQQQPQ) and to 11–19 were digestedin vitro with small intestinal mucosa from children with celiac disease in remission and from normal children. The products of digestion were separated into two fractions on the basis of M_r<400 and M_r>400 by gel permeation chromatography and subjected to amino acid analysis. After digestion of the dodecapeptide with celiac mucosa, 71±14% (molar) of the total digestion products remained in the M_r>400 fraction. Glutamine, proline, serine, and asparagine were the major amino acids present. Glutamine, proline, and leucine were the major amino acids in the M_r<400 fraction. The M_r>400 fraction from the celiac mucosal digestion of the nonapeptide was of similar composition to the corresponding fraction from the dodecapeptide and represented 78±15% of the total products. Digestion of the two peptides with normal mucosa gave lower amounts of products in the M_r>400 fraction, but they were of similar composition to the corresponding fractions from the celiac mucosal digestion. Peptides such as NPSQQQP and QNPSQQQ may be present in the M_r>400 fractions since glutamine and proline are present in the approximate ratio of 21, respectively. The results indicate a defect in the mucosal digestion of peptides which are active in an animal model of celiac disease.     

Partial In Vitro Digestion of Active Gliadin-Related Peptides in Celiac Disease

Peptides corresponding to residues 75–86 (RPQQPYPQPQPQ) and 75–85 of the A-gliadin structure, which were shown to be active in an animal model of celiac disease, were digested in vitro with small intestinal mucosa from children with celiac disease in remission and with mucosa from normal children. The products of digestion were separated into two fractions by gel permeation chromatography. Undigested residues (M _r > 400 fraction) from both peptides contained mainly glutamine, proline, and tyrosine, while the digested materials (M _r < 400 fraction) contained mainly proline, glutamine and arginine. Much larger amounts of undigested peptides were obtained from digestion with celiac mucosa than from normal mucosa. The results with peptide 75–86 indicated that the octapeptide 77–84 (QQPYPQPQ) was the main residual component and this peptide was shown to be active in the assay. Peptide 77–84 was also obtained as a residue from digestion of peptide 75–85, together with heptapeptide 77–83. The results lend further support for a primary mucosal defect in celiac disease and indicate that residual peptides in the small intestine of patients with the disease still retain appreciable toxicity.     

Partial In Vitro Digestion of Active Gliadin-Related Peptides in Celiac Disease

Peptides corresponding to residues 75–86 (RPQQPYPQPQPQ) and 75–85 of the A-gliadin structure, which were shown to be active in an animal model of celiac disease, were digested in vitro with small intestinal mucosa from children with celiac disease in remission and with mucosa from normal children. The products of digestion were separated into two fractions by gel permeation chromatography. Undigested residues (M _r > 400 fraction) from both peptides contained mainly glutamine, proline, and tyrosine, while the digested materials (M _r < 400 fraction) contained mainly proline, glutamine and arginine. Much larger amounts of undigested peptides were obtained from digestion with celiac mucosa than from normal mucosa. The results with peptide 75–86 indicated that the octapeptide 77–84 (QQPYPQPQ) was the main residual component and this peptide was shown to be active in the assay. Peptide 77–84 was also obtained as a residue from digestion of peptide 75–85, together with heptapeptide 77–83. The results lend further support for a primary mucosal defect in celiac disease and indicate that residual peptides in the small intestine of patients with the disease still retain appreciable toxicity.     

Transformation of macromolecules from a brown coal by lignin peroxidase

Indirect evidence has suggested that lignin peroxidase (LiP) of the white-rot fungus Phanerochaete chrysosporium catalyses oxidative decolourisation and depolymerisation of macromolecules from brown coal in vivo. In this study we show that LiP catalyses these transformations in vitro. Unmethylated (USC45 coal) and methylated (MWSC6 coal) fractions of solubilised macromolecules (M _r > 30 000) from a brown coal were treated with a semi-purified preparation of LiP isozymes from P. chrysosporium. Both coal fractions were decolourised, losing between 26% and 39% of their absorbance at both 280 nm and 400 nm, in reactions that had an absolute requirement for H₂O₂ and veratryl alcohol. Neither coal fraction was transformed when the enzyme was heat-inactivated or in the presence of the LiP inhibitor metavanadate. Gel-permeation chromatography showed that MWSC6 coal but not USC45 was depolymerised and yielded low-molecular-mass (M _r < 30 000) fragments. Nine monomeric products were identified by GC-MS. Received: 20 March 1998 / Received revision: 3 September 1998 / Accepted: 3 September 1998     

The effect of fungal laccase on fractionated lignosulphonates (peritan Na)

Two fractions obtained after chromatography of lignosulphonates on Sephadex G-50, varying in M_rs, were treated with extracellular Trametes versicolor laccase. After incubation of the low M_r fraction, polymerization was observed, while in the case of the high M_r fraction the reverse process occurred. As a result of depolymerization, five new lower M_r fractions appeared. The reaction reached peak level after 2 hr of incubation and then the quantities of the products diminished, possibly due to their repolymerization. These studies indicate that laccase possesses both polymerization and depolymerization activity though the former was predominant.     

Specific non-histone protein fractions associated with ‘active’ chromatin in immunized rats

Summary Non-histone proteins of normal, non-immunized rats and rats immunized with mouse spleen cells were labelled with three different amino acids: [³H]tryptophan, [³H]methionine and [³H]leucine. Chromatin was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and residual. Non-histone protein fractions F (M_r 12 000) and H (M_r 3 000) highly labelled with [³H]tryptophan, lower with [³H]methionine but not with [³H]leucine, were present mainly in the residual fraction. After DNAse II treatment non-histone protein fractions F and H disappeared in chromatin fractions and were present in Mg^2– soluble fractions which suggests that, similar to the fractions I (M_r below 3 000) and B (M_r 120 000) described previously (5), these fractions may be associated with active transcribed genes.     

A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization

We previously described the purification and characterization of a 37,000 M_r cysteine proteinase, designated EP-A, from gibberellic acid (GA₃)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M_r of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M_r as determined by gradient SDS-PAGE.     

Identification of new peptides synthesized under the hydrogenase control system of Alcaligenes eutrophus

Total protein of Alcaligenes eutrophus was analyzed by two-dimensional protein map. Cells grown at 30° C expressed hydrogen-oxidizing (Hox) ability mediated by a soluble (Hos) and a particulate hydrogenase (Hop). Hox ability was not expressed at 37° C (HoxTs). The six subunits of the two hydrogenases were identified. Besides these six subunits eight peptides were not or hardly detected at 37° C. The mutant HF117 which expressed Hox ability at 37° C (HoxTr), formed the hydrogenase peptides and five of the eight peptides. These peptides designated B, C, E, F, and H were characterized by their isoelectric point and molecular mass (M _r); their M _r were 18 800, 45 400, 41 900, 39 400, and 40 600, respectively. The five peptides were not formed in regulatory Hox^– mutants, and not formed in mutants cured of plasmid pHG1, carrying the genetic information for hydrogenase formation. Strain HF160, carrying a Tn5 insertion in a gene essential for Hos expression specifically did not form the B-peptide. All peptides were found in the soluble fraction of cell extracts, the F-peptide was also detected in the particulate fraction. The function of the new Hox-peptides is presently unknown.Abbreviations PAGE polyacrylamide gelelectrophoresis - SDS sodium dodecylsulfate - Hox hydrogen oxidizing ability     

Partial characterization of immunosuppressive proteins from bovine uterine luminal secretions

Unfractionated and fractionated uterine luminal protein (ULP) secretions collected from nonpregnant and pregnant beef cows on Day 17 post-breeding were tested in vitro for suppression of lymphocyte blastogenesis to the mitogen phytohemagglutinin (PHA). In replicated experiments, ULP from nonpregnant and pregnant cows was separated into five molecular weight (M_r) fractions with Sephacryl S-200. Unfractionated (25 to 400 μg/ml) and fractionated (25 to 100 μg/ml) ULP was added to cultures containing 5 × 10⁵ bovine lymphocytes and 0.4 μg of PHA in a complete culture medium. At 48 hr, 0.5 μCi of ³H-thymidine was added to cultures, and cells were harvested at 60 h by automation. Thymidine incorporation data were expressed as percentage of control (no ULP) values. Unfractionated and all S-200 ULP fractions from nonpregnant and pregnant cows suppressed (P<0.05 to 0.001) lymphocyte blastogenesis to PHA, but to varying degrees of suppression. Unfractionated ULP was more suppressive (P<0.05) for pregnant than nonpregnant cows, which was likely due to the greater (P<0.05) immunosuppressive activity of S-200 fractions I (>219,000 M_r) and V (∼14,000 M_r) from the pregnant cows. At 25 μg ULP/ml, mean (± SEM) % of control values for fraction I from pregnant and nonpregnant cows were 9.1 ± 3.3 and 36.6 ± 8.5%, respectively (P<0.05). Values for fraction V were 15.7 ± 6.5 and 46.6 ± 6.1%, respectively (P<0.01). Within each reproductive class, fractions I and V were more suppressive (P<0.05) than fractions II, III and IV. Immunosuppression was not mediated by lymphocyte cytotoxicity.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l⁻¹ exopolysaccharides with glucose and 30 mg l⁻¹ with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M _r) of 1.7 × 10⁶ and 4 × 10⁴ in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M _r of 4 × 10⁴. The high-M _r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M _r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M _r fractions appeared to be dependent on the carbohydrate source, whereas the low-M _r fractions were produced more continuously. Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997     

Computer analysis identifies sequence homologies between potential gene products of Maize Streak Virus and those of Cassava Latent Virus and Tomato Golden Mosaic Virus

Summary The amino acid sequences of the putative polypeptides of maize streak virus (MSV) have been systematically compared with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) using the programme DIAGON (8).Conserved sequences have been detected between peptides encoded by the complementary (-) sense of MSV and those of CLV and TGMV, viz; the 40 200 M_r polypeptide of CLV-1 (3) and the 40 285 M_r polypeptide of TGMV-A (4) show extensive homologies with the 17 768 M_r and 31 388 M_r polypeptides of MSV (6).Distant and variable homologies have been detected between the putative coat protein of MSV when compared with those of CLV and TGMV. No other relationships between the potential gene products of MSV and those of CLV and TGMV have been detected.The extensive homologies detected between the complementary sense encoded peptides suggest that they are derived from functional genes, and that the directly conserved sequences may contain amino acids essential to the function of these proteins. The less extensive homologies among the putative coat proteins are considered in relation to their possible structures and functions.     

Enzyme induction in soybean infected by Phytophthora megasperma f.sp. glycinea

Laminin, the glycoprotein of basement membranes, consists of two subunits of 200,000 (α) and 400,000 (β) M_r on gel electrophoresis after reduction. We evaluated the relative proteolytic susceptibility of the two subunits using a variety of serine proteases. Human α-thrombin degraded the β subunit without altering the density or size of the α subunit. Chymotrypsin, plasmin, and cathepsin G all degraded both the β and α subunits producing limited digestion products. Chymotrypsin and cathepsin G both produced two major fragments of 160,000 and 130,000 M_r whereas plasmin produced two fragments of 180,000 and 140,000 M_r. Time course digestion studies demonstrated that the 400-kd β subunit was digested much more rapidly than the α subunit, and suggested that the major fragments (greater than 100,000 M_r) produced by chymotrypsin, plasmin, and cathepsin G were derived from the α subunit. The latter supposition was confirmed by first digesting laminin with thrombin to completely remove the β subunit, followed by digestion with chymotrypsin, cathepsin G, or plasmin. We conclude that the β subunit of laminin is highly protease labile. In contrast, the α subunit contains a large region resistant to serine proteases. Electron microscopic studies of the purified fragment of laminin derived from digestion with cathepsin G demonstrated that the protease resistant region of the α subunit contained three arms of similar appearance (32 nm) and included the intersection of the three short arms of the laminin molecule.     

Amino acid sequences of three acyl-binding/lipid-transfer proteins from rape seedlings

The complete amino acid sequence of three acyl-binding/lipid-transfer proteins, AB/LTP I, AB/LTP II and AB/LTP III from germinated rape seeds were determined. AB/LTP I and AB/LTP II consist of 93 residues and the M_r was determined as 9408 by mass spectrometry and calculated as 9406.8 from the sequence. AB/LTP III consists of 92 residues and the M_r was determined as 9424 by mass spectrometry and calculated as 9422.8 from the sequence. The primary structures were determined by automated Edman degradations of the intact proteins and peptides obtained from digestion with trypsin and endoproteinase Asp-N and cyanogen bromide cleavage. Use of ²⁵²Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides.     

Spatial organization of digestion,secretory mechanisms and digestive enzyme properties in Pheropsophus aequinoctialis (Coleoptera: Carabidae)

Aminopeptidase (soluble form M_r 110,000), carboxypeptidase A (soluble form M_r 47,000), maltase (a dimer composed of two identical M_r 60,000 subunits) and trypsin (two charge isomers with M_r 34,000) are found in major amounts in the crop and midgut tissue, whereas amylase (a trimer of three identical M_r 18,000 subunits) and cellobiase (a trimer of three identical M_r 27,000 subunits) occur mainly in the crop and midgut contents. Subcellular fractions of midgut cells were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation. The results suggest that part of the aminopeptidase and carboxypeptidase A activity is bound to microvilli, that major amounts of trypsin and maltase are trapped in the cell glycocalyx and finally that soluble aminopeptidase, amylase and cellobiase occur in intracellular vesicles. The data support the hypothesis that most protein and carbohydrate digestion takes place in the crop under the action of enzymes passed forward from the midgut, after being secreted by exocytosis. Nevertheless, part of the intermediate and final digestion occurs at the surface of the midgut cells. The peculiar features of the digestion of P. aequinoctialis beetles, including their partly fluid peritrophic membranes, are thought to be derived from putative Coleoptera ancestors.     

Characterization of protective antigens from the midgut of Amblyomma variegatum ticks

Separation of midgut membrane proteins from the tick,Ambylomma variegatum, using a non-ionic detergent (TritonX-114), resulted in two protein fractions, namely DET (detergent) and AQ (aqueous). In immunoblotting analysis with polyclonal antibodies against these fractions, 4 proteins (M_r ∼27,000, 67,000, 86,000 and 95,000,)and 2 proteins (M_r ∼54,000 and 67,000) were detected in the DET and AQ fractions, respectively. Three of the DET fraction proteins M_r∼27,000, 67,000 and 95,000 were glycosylated since they bound to the lectin, concanavalin A. In 2-dimensional gel electrophoresis, the AQ and DET fraction proteins were found to be acidic in nature. In a series of bioassay experiments, rabbits were first immunised with both DET and AQ fractions and then infested with ticks. The egg batch weights of these ticks were reduced by 50% compared to control ticks. Furthermore, there was a significant reduction in the hatchability of eggs laid by ticks fed on rabbits previously immunised with both DET (14%) and AQ (33%) fractions. Based on the egg hatchability, there productive capacity of ticks was reduced by 77 and 48% by DET and AQ fractions, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Peptide mapping of contractile proteins: Two-dimensional analysis of cyanogen bromide fragments on polyacrylamide gels

Peptide mapping of contractile proteins is necessary for correlation of the structual properties of these molecules with their distinct roles in various cell types. The large size of several of these polypeptides requires their cleavage by cyanogen bromide and the separation of the resulting products on a two-dimensional polyacrylamide-gel system to ensure reasonably complete peptide maps. Such a peptide map of rabbit skeletal muscle actin is in agreement with predictions from the amino acid sequence. The peptide map of rabbit skeletal muscle myosin can be resolved into maps of heavy and light meromyosins resulting from limited tryptic digestion of the myosin. Unique regions of the myosin map are occupied by one or the other of these segments. Analysis of native 105,000 M_r elam adductor paramyosin and its 94,000 M_r proteolytic products indicates that specific changes in peptide composition have occurred.     

The peptides of chloroplast membranes: I. The soluble coupling factor (Ca2+-ATPase)

The chloroplast coupling factor (CF₁) was analyzed by gel electrophoresis in SDS and found to contain two major bands in equal amounts with mobilities corresponding to molecular weights of 62,000 and 57,000 and three minor bands of molecular weights 38,000, 21,000, and 14,000. The peptides were present in comparable amounts in many different preparations of the protein and, therefore, were thought not to be tightly bound contaminants. The interaction between these five peptides was shown to be noncovalent.Incubation of the enzyme with trypsin, under conditions which activate the latent ATPase, was found to cause selective digestion of the five peptides; the 62,000 M_r peptide was the most susceptible to digestion, while the 57,000 M_r peptide was most stable to trypsin. When chloroplast membranes were exposed to trypsin in the light to activate the postillumination Mg²⁺-dependent ATPase activity, EDTA extraction solubilized a protein fraction which contained the normal CF₁ peptide pattern. Also, the membranes, when solubilized and chromatographed on SDS-gels did not show the disappearance of any band.The ATPase activity of the protein was highly susceptible to ionic strength, being 50% inhibited by monovalent salts at a concentration of 0.05 m.     

Characterization of a cDNA encoding the protein moiety of a putative arabinogalactan protein from Lycopersicon esculentum

The nucleotide sequence of a cDNA prepared from poly(A)⁺ RNA from Lycopersicon esculentum fruit codes for a protein, M _r 20812, with features representative of the protein core of arabinogalactan proteins. The deduced amino acid sequence resembles that of peptides of arabinogalactan proteins isolated from carrot and rose and is most similar to the sequence of tryptic peptides from Lolium multiflorum (Gleeson et al., Biochem J 264 (1989) 857–862). The similar sequences include a number of Ala-Pro repeats, a feature considered distinctive of arabinogalactan proteins. The amino acid composition is similar to that of the peptide core of the Lolium multiflorum arabinogalactan protein; alanine, serine and proline account for 57% of the polypeptide. The mRNA corresponding to the cDNA sequence was detected in roots, leaves and fruit. The levels of mRNA are reduced in older leaves, in fruit that have commenced ripening and in leaves and fruit that have been wounded.     

Characterization of fragments produced by clostripain digestion of proteoglycans from the Swarm rat chondrosarcoma

Rat chondrosarcoma proteoglycan aggregate samples were digested with the protease clostripain (from Clostridium histolyticum) for various times. The progress of digestion was studied by Sepharose 2B chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After complete digestion, the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid was separated from the chondroitin sulfate-peptide clusters released by the enzyme. In this limit complex, the M_r of the link protein was 42,000, slightly smaller than the M_r of 45,000 observed for intact link protein. The chondroitin sulfate-peptides contained an average of about seven to eight polysaccharide chains per peptide and, after chondroitinase ABC digestion, were found to consist of two size classes of peptides. By comparison, chondroitin sulfate-peptides isolated from trypsin digests contained four to five chains per peptide and contained primarily the smaller size class of peptides. At early digestion times with clostripain, several distinct molecular weight intermediates containing hyaluronic acid-binding sites were identified on sodium dodecyl sulfate-polyacrylamide gels. These intermediates, with M_r, values of about 125,000, 100,000, and 85,000, decreased with increasing digestion time to yield a limit polypeptide (M_r = 67,000). Procedures are described for purifying this limit polypeptide and the link protein for further characterization. The results indicate that clostripain can be used to fragment proteoglycan molecules selectively to define different functional regions for study.