Major Digestion Products Research Articles

Abstract 5684: B-catenin-dependent and independent changes in gene expression induced by sphingolipids

Abstract Dietary supplementation with sphingolipids inhibits colon tumorigenesis in carcinogen-treated CF1 mice and in multiple intestinal neoplasia (Min) mice that produce intestinal tumors spontaneously. In both cancer models the regulation of β-catenin by sphingolipid metabolites is an important early event. Stabilization and nuclear translocation of β-catenin and its interaction with T-cell factor (TCF)/lymphoid enhancer factor (LEF) play important roles to activate the transcription of genes associated with cancer development and progression. Sphingosine and ceramides, major digestion products of complex sphingolipids in the diet, down-regulate β-catenin transcriptional activity in a dose- and time-dependent manner. Our data indicate that sphingosine and ceramides suppress SW480 human colon cancer cell growth in a concentration-dependent manner. In the present study, we investigated the dependency of sphingosine- and ceramide-induced changes in gene expression on β-catenin transcriptional activity by comparing control and β-catenin siRNA treated cells. We observed changes in the expression of genes involved in signal transduction, transcription factors, cell cycle control and DNA damage repair, cell adhesion, cell invasion and metastasis, angiogenesis, and apoptosis and cell senescence. Most genes were regulated by all metabolites; however, some genes were only targeted by specific metabolites. Furthermore, only a small number of genes regulated by the sphingolipid metabolites relied completely on the activation of the β-catenin transcriptional activity. Further experiments are currently being performed to confirm the function of β-catenin on the regulation of these genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5684.

Enzyme-assisted extraction of arsenic species from plant material

Investigations regarding the transfer and metabolism of arsenic species in plants require mild extraction conditions to conserve the original composition of arsenic species. Beside the use of water or water/methanol for extraction of arsenic species from plant samples, enzymes can assist this procedure by digestion of cellulose and other constituents of cell walls, resulting in a faster, more efficient extraction technique which preserves the arsenic species. The investigations presented here were focused on the stability of certain arsenic species in enzymatic solutions, optimal conditions for their chromatographic separation and detection namely by means of ion chromatography–inductively coupled plasma mass spectrometry and improvements with respect to extraction efficiency. With commercially available enzymes and enzyme mixtures, the digestion rate of soluble starch as model cellulose was determined using high-performance anion exchange chromatography–pulsed amperometric detection analysis of glucose as the major digestion product. The most effective digestion rate (80% within 4 h) was obtained with Viscozyme®. This enzyme mixture was applied to extracted arsenic species from algae and terrestrial plant materials. Qualitative and quantitative differences in the results between enzyme-assisted and water extractions were obtained and discussed. The results show that the application of enzymes in mild extraction protocols should be evaluated as an additional step for the identification of As-metabolics in organisms. Careful selection of suitable enzyme mixtures can overcome the disadvantage that extraction efficiency is very organism-specific.

Digestion of plant monogalactosyldiacylglycerol and digalactosyldiacylglycerol in rat alimentary canal

We investigated digestion of orally fed galactoglycerolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) from wheat flour in the rat alimentary canal, especially focusing on the digestive fates of deacylated galactosylglycerol structures. After a single oral administration of MGDG (20 mg/rat), monogalactosylmonoacylglycerol and monogalactosylglycerol (MGG) were found to be major digestion products in the intestinal tract. Similarly, digalactosylmonoacylglycerol and digalactosylglycerol (DGG) were confirmed to be present in the intestinal tract after DGDG ingestion (20 mg/rat). In rats fed wheat flour glycolipids (42 mg MGDG and 81 mg DGDG per rat), completely deacylated galactosylglycerols (MGG and DGG) were not detected in portal plasma. Although the deacylated galactosylglycerols were not significantly decomposed by intestinal mucosa in vitro, they were hydrolyzed by cecal contents. The results demonstrated that orally ingested plant galactoglycerolipids in the rat alimentary canal are rapidly hydrolyzed into constituent fatty acids and that hydrophilic galactosylglycerols and the hydrophilic backbone galactosylglycerols are not absorbed from intestine or degraded into galactose and glycerol in the intestinal tract. Therefore, the presence of deacylated galactosylglycerols may affect the fermentative activity of enterobacteria in the cecum and colon.

Effector-induced self-association and conformational changes in the enhancer-binding protein NTRC.

The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of sigma N-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATP gamma S concentration suggested an apparent dissociation constant (Kd) for ATP gamma S of 150 microM. In the presence of DNA, both the central and C-terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change. NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NTRC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NTRC displayed a monomer-dimer equilibrium with a Kd of 6 microM. In the presence of ATP gamma S the equilibrium was shifted towards the dimer form (Kd = 0.8 microM). A similar dissociation constant for the monomer-dimer interaction was observed with NTRC S160F in the absence of ATP gamma S (Kd = 0.5 microM). The addition of ATP gamma S induced a significant association of NTRC S160F to higher-order states with a dimer-octamer model producing a slightly, but not significantly better fit to the data than a dimer-hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Structure Characterization of Sulfate and Oxysulfate Phases Formed During Sulfuric Acid Digestion of Ilmenites

Laboratory digestion of natural and upgraded ilmenites with concentrated sulfuric acid has been carried out under conditions simulating the sulfate-route pigment process. X-Ray diffraction studies on the solid digestion cakes led to the characterization of a number of different iron titanium sulfate and oxysulfate compounds, whose formation was dependent on the ilmenite composition and digestion conditions. Two different sulfate phases were identified, with structures related to those for Fe2(SO4)3 and H2O[ Zr (HPO4)2]. Four different iron titanium oxysulfates , ( Fe,Ti )(O,OH)SO4, were identified as major digestion products, three having structures related to those for β-NbOPO4, GeOHPO4 and lazulite , Mg[AlOHPO4], and the fourth being a new struture type. The structures of the oxysulfate phases were refined by the Rietveld method. Both the sulfates and the oxysulfates have three-dimensional framework structures formed by corner linking of ( Ti,Fe )O6 octahedra and SO4 tetrahedra, and containing channels or interlayer regions that can be occupied by cations such as H3O+ and Fe2+. The different structures have many features in common. Their structural relationships are discussed and mechanisms are proposed for the phase transformations encountered in the digestion studies.

Biochemical and Functional Characterization of a Recombinant GTPase, Rab5, and Two of Its Mutants

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

The 84-kDa form of human matrix metalloproteinase-9 degrades substance P and gelatin.

Matrix metalloproteinase-9 (MMP-9) is secreted from cells and, once activated, is thought to degrade collagen in the extracellular matrix. Because collagen is not readily localized where neurons have been shown to produce MMP-9 in the human brain, the ability of this enzyme to degrade bioactive peptides was investigated with representative tachykinin peptides [substance P (SP), neurokinin A, neurokinin B, and kassinin]. Latent MMP-9 (94 kDa) was purified from the human cell line HL-60 and converted to an intermediary active form (84 kDa) with p-aminophenylmercuric acetate. This active form of MMP-9 degraded SP with a kcat/Km of 170 mM-1 min-1, which is 30-fold greater than the previously reported value for a representative collagen-derived peptide. The major digestion products were identified as SP and SP, which were derived from cleavage of the Gln6-Phe7 bond. Minor products were also generated from cleavage of the Gly9-Leu10 bond. The other representative tachykinin peptides were cleaved at rates > 10-fold slower than that of SP. The 84-kDa peptidase was also active as a gelatinase. Longer treatment of MMP-9 with p-aminophenylmercuric acetate caused the conversion of the 84-kDa enzyme to the established 68-kDa active form; however, the rate of SP degradation did not increase. Because MMP-9 is produced by neurons of the CNS, these results suggest a possible regulatory role for the enzyme in interacellular communication by altering the availability of bioactive peptides.

The TyrR protein of Escherichia coli, analysis by limited proteolysis of domain structure and ligand-mediated conformational changes.

The TyrR protein of Escherichia coli K12 is a homodimer containing 513 amino acids/subunit. This protein is important in the transcriptional regulation of several genes whose protein products catalyze steps in aromatic amino acid biosynthesis or transport. Methods were developed for efficiently purifying the TyrR protein to apparent homogeneity. We analyzed the pattern of cleavage of the TyrR protein by trypsin, either in the absence of ligands or in the presence of saturating levels of L-tyrosine, ATP, or poly(dI-dC). At low (1:200 ratio by weight) trypsin levels, in the absence of ligands, two major digestion products accumulated. These were polypeptides of 22 and 31 kDa, shown to contain amino acid residues 1-190 and 191-467, respectively. The pattern of trypsin cleavage was unaffected by tyrosine. In the presence of ATP, an intermediate species of 53 kDa, probably containing amino acid residues 1-467, was observed. The kinetics of appearance of the 53-kDa species were consistent with a role for ATP in accelerating the hydrolysis of the R467-F468 peptide bond. The 53-kDa polypeptide underwent further tryptic hydrolysis to yield fragments of 22 and 31 kDa. When both tyrosine and ATP were present, the rate of formation of the 22- and 31-kDa fragments was more rapid than in the absence of these ligands. It appears that when both ligands are bound, the rates of hydrolysis of peptide bonds R190-Q191 and R467-F468 are both enhanced. Additional limited proteolysis experiments suggested that polypeptide segment 191-467 contains ATP binding site(s), and that the rate of cleavage of peptide bonds R190-Q191 and R467-F468 is altered when the TyrR protein interacts with poly(dI-dC), an analog of target DNA. Our results reveal the presence of two major structural domains within the TyrR protein. The first domain (amino acid residues 1-190) is extremely resistant to hydrolysis by trypsin. The second domain (residues 191-467), which is likely to contain ATP-binding site(s), is homologous to several other transcriptional activators specific for promoters responsive to the sigma 54 form of RNA polymerase. The remainder of the TyrR protein (residues 468-513) contains the operator recognition elements, probably arranged in the form of a helix-turn-helix motif. This polypeptide segment was not detected as a discrete tryptic hydrolysis product.

The heterogeneity of bovine IgG2—IV. Structural differences between IgG2a molecules of the A1 and A2 allotypes

The major product of digestion of bovine IgG2a(A1) with immobilized pepsin is F(ab') 2 while similar treatment of IgG2a(A2) yields Fab, Fc and other products. It is postulated that structural differences in the hinge region, including the absence of the most N-terminal disulfide bridge in IgG2a(A2) and a displacement of the primary pepsin cleavage site toward the Fd, can explain this effect. The immunodominant A1 allotope(s) appears to be located in the CH3 domain of IgG2a(A1) while the A2 allotopes are located elsewhere and are apparently affected by digestion. The allotypic bias of rabbit anti-IgG2a is also present in anti-IgG2a raised in goats. However, the A2 allotypic determinants of bovine IgG2a are recognized by goat precipitins although precipitins of this specificity are not detectable in rabbits immunized with IgG2a(A2). Rabbit anti-A2 antibodies are detectable using the ELISA in rabbits immunized with IgG2a(A2).

A novel endo-β(1,4)-d-glucuronidase activity inMucor rouxii

Extracts from the fungus Mucor rouxii contain an enzyme activity which can degrade the homopolymeric β(1,4)-d-glucuronan (mucoric acid) of the fungal wall. The enzyme can also degrade the homopolymeric glucuronan domains of mucoran, a complex polyuronide of the Mucor wall. The enzyme is active at neutral pH and has an estimated Mr of 27 500. The major product of digestion is a disaccharide, suggesting an endolytic mechanism.

Complementation of human growth hormone (GH) peptide 1-134 with C-terminal fragments of human GH produced by digestion with bromelain.

The digestion of human GH (hGH) with the proteolytic enzyme, bromelain, results in a major product consisting of a mixture of three large fragments, i.e. residues 1-135 + 143-191, 1-135 + 145-191, and 1-135 + 146-191. In the case of each fragment, the N-terminal peptide is joined to the C-terminal fragment by the disulfide bridge between residues 56 and 165. A C-terminal fragment mixture consisting of peptides 143-191, 145-191, and 146-191 was isolated from this major digestion product after reduction and S-carbamidomethylation of its disulfide bonds. In the present study, the noncovalent complementation of the peptides in this mixture with S-carbamidomethylated peptide 1-134 derived from thrombin-digested hGH was investigated. Noncovalent complementation of these peptides was accomplished by dissolving equimolar amounts of the materials in 0.5% ammonium bicarbonate-6 M guanidine-HCl and dialyzing the mixture slowly to remove the guanidine-HCl. The recombinant mixture was recovered in 26% yield by gel filtration of the peptide mixture and was found to contain three noncovalent recombinant species, i.e. peptides 1-134 + 143-191, 1-134 + 145-191, and 1-134 + 146-191. Thus it would appear that residues 135-145 are not required to obtain noncovalent complementation between the N- and C-terminal regions of the hGH molecule. In an RIA for hGH the recombinant mixture was found to possess approximately 40% the cross-reactivity of the native hormone. In contrast, it had only about 10% the activity of native hGH in the weight gain test in hypophysectomized rats, in stimulating phenylalanine incorporation into the protein of the isolated hypophysectomized rat diaphragm, and in stimulating glucose oxidation by isolated adipose tissue of hypophysectomized rats. The limited biological activity of the recombinant mixture is of interest, since the major bromelain digestion product from which the C-terminal peptides were derived consists of a mixture of rather similar molecules (i.e. peptides 1-135 + 143-191, 1-135 + 145-191, 1-135 + 146-191; and with intact disulfide bridges), which exhibits substantial growth-promoting and insulin-like activities.

Fragments of human growth hormone produced by digestion with bromelain: Chemistry and biological properties

In an effort to produce small discrete fragments of human growth hormone (GH), we examine the action of the proteolytic enzyme, bromelain, on this molecule. Purified human GH incubated for 40 min at 22°C with crude bromelain and gel-filtered on Sephadex G-100 resulted in a major digestion product, peak 2. SDS-urea gel electrophoresis in the presence of β-mercaptoethanol suggested that peak 2 was composed of two polypeptide chains. Two polypeptide fractions were isolated by the reduction and S-alkylation of peak 2 in 6 M guanidine-HCl and subsequent chromatography on Sephacryl S-200 in 6 M guanidine-HCl. These two fractions, A and B, had the same mobilities as the two components of peak 2 on SDS-urea gels. Amino-terminal analysis, tryptic peptide mapping, carboxypeptidase digestion, cyanogen bromide cleavage, and amino acid analysis of fraction A indicated that it was peptide 1–135. Amino-terminal analysis and tryptic peptide mapping of fraction B suggested the presence of a mixture of peptides 143–191, 145–191 and 146–191. Thus, peak 2 is heterogeneous and appears to be a mixture consisting of peptide 1–135 + peptide 143–191, peptide 1–135 + peptide 145–191 and peptide 1–135 + peptide 146–191, in each case the N-terminal peptide being joined to the C-terminal peptide by the disulfide bridge between residues 53 and 165. In the weight-gain test in hypophysectomized rats, two preparations of peak 2 appeared to be somewhat less active than the native human GH preparations from which they were derived. Several preparations of peak 2 showed equivalent potency in stimulating [ 14C]glucose oxidation to 14CO 2 by isolated epididymal adipose tissue of hypophysectomized rats. Also, most of the peak 2 preparations were somewhat less active than native human GH in displacing 125I-labeled human GH bound to antibodies to human GH.

Partial Tryptic Digestion of Cucurbitin from Pumpkin Seed

A stable digestion intermediate was observed when cucurbitin, the crystalline storage globulin of pumpkin [Cucurbita moschata (Duch.) Poir.] seed, was treated with trypsin. The major digestion product, termed cucurbitin-T, was isolated as a homogeneous protein of M, 285 000+ 10000 by chromatography over Sepharose CL-6B. The treatment with trypsin lowered the isoelectric point, decreased the charge heterogeneity, and increased the solubility of cucurbitin. Electrophoresis of denatured and reduced cucurbitin-T in 8 M urea at pH 8.5 suggested that the basic polypeptide chains of cucurbitin were degraded less by the enzyme treatment than were the larger acidic chains. This difference in susceptibility was confirmed by two-dimensional electrophoresis. The amino acid analyses of cucurbitin and cucurbitin-T subunit polypeptides are consistent with the changes demonstrated by electrophoresis. It is suggested that the larger fragments (M, 20000-24000) of the acidic chains found in cucurbitin-T, together with the more resistant basic chains (M, - 22000) in both forms of cucurbitin, may constitute the 12 equivalent structural domains observed for cucurbitin by X-ray diffraction analysis.

Reactions of erythrocyte glycoproteins and their degradation products with various anti-I sera.

Three fractions of erythrocyte glycoproteins obtained from Sepharose 4-B chromatography were tested for I activity with ten serologically differentiated anti-I sera. The most active was fraction I, eluted at the void volume and containing the lowest amount of alkali-labile oligosaccharide chains. The desialization of glycoproteins increased their activity toward anti-I-s and anti-I-D sera, and did not change or decreased the activity toward anti-I-F sera. The most abundant fraction II (major sialoglycoprotein of erythrocyte membranes) showed no or only a very weak I activity, but I-active glycopeptides were isolated from products of digestion of fraction II with trypsin. The major product of digestion, sialoglycopeptide IIT-2 showed I activity only after alkaline elimination of alkali-labile oligosaccharide chains. The results indicate that I receptors are present in hindered form on apparently I-inactive components of erythrocyte membrane.

Lysozyme-catalyzed Hydrolysis and Transglycosylation Reactions of Bacterial Cell Wall Oligosaccharides

Tritium-labeled tetrasaccharide, GlcNAc-β(1 → 4)-MurNAc-β(1 → 4)-GlcNAc-β(1 → 4)-MurNAc, and disaccharide, GlcNAcβ(1 → 4)-MurNAc (where MurNAc represents N-acetylmuramyl), were prepared by tritiation of the corresponding unlabeled compounds. The products, obtained in good yield, were found to be homogeneous and radiochemically pure by several chromatographic and electrophoretic criteria and indistinguishable from the starting materials. Chemical and enzymatic studies indicated that they were uniformly labeled. The action of lysozyme on the tetrasaccharide was studied by a combination of paper electrophoresis and paper chromatography. The major product of digestion was the disaccharide. Higher oligosaccharides were formed in the initial stages of the reaction and disappeared after prolonged incubation when only disaccharide together with considerable amounts of tetrasaccharide was present. The rate of disappearance of the tetrasaccharide changed markedly during the course of the reactions; the maximal rate increased with the increase in substrate concentration. Four higher oligosaccharides (hexa-, octa-, deca-, and dodecasaccharides) were isolated from lysozyme digests of the tetrasaccharide-3H. Two of these were found to be identical with the hexa- and octasaccharides isolated from lysozyme digests of Micrococcus lysodeikticus cell walls. The higher oligosaccharides were readily digested by lysozyme to yield the corresponding di- and tetrasaccharides. The hexa- and octasaccharides were digested at a much faster rate than the tetrasaccharide. Addition of small amounts of octasaccharide to the tetrasaccharide increased significantly the rate of digestion of the latter. Upon incubation of unlabeled tetrasaccharide with disaccharide-3H in the presence of lysozyme, the labeled tetrasaccharide formed did not contain significant amounts of radioactivity in the nonreducing half of the molecule. Furthermore, when this newly formed radioactive tetrasaccharide was incubated with GlcNAc-14C and lysozyme, the Glc-NAc-β(1 → 4)-MurNAc-β(1 → 4)-GlcNAc contained insignificant amounts of 3H. These results indicate that in the reactions studied over 90% of the new products must be formed by transglycosylation, and not by reversal of hydrolysis. The results obtained show that hydrolysis of the tetrasaccharide to disaccharide proceeds chiefly via transglycosylation, leading to the formation of high oligosaccharides. This mechanism is consistent with the binding data for the association of various saccharides with lysozyme and can be readily explained on the basis of the three-dimensional model of lysozyme presented by Phillips and co-workers.