Purification of a new intestinal anti-proliferative factor from normal human small intestine.

Previous studies suggest that intestinal cell proliferation may be controlled by endogenous mitosis inhibitors. We describe here the isolation of a protein named intestinal anti-proliferative factor (IAF) from human small intestine. Successive DEAE anion exchange, isoelectric focusing and gel filtration chromatographies led to a purified anti-proliferative protein fraction used to produce antibodies. Using these antibodies as affinity chromatography ligand, IAF was purified from human small intestine cytosolic fraction. IAF was a potent inhibitor of adenocarcinoma colon cells (HT-29 D4 line) DNA synthesis and proliferation with 50% inhibition observed at picomolar concentrations. Analyzed on SDS/PAGE under reducing conditions, this protein migrates with an apparent molecular mass of 120 kDa and amino acid sequence of two internal peptides displays no homology with another listed protein. Cell cycle studies showed that the growth inhibitory effect was maximal between mid G1 and early S phases. Moreover, flow cytometry studies demonstrated that IAF inhibited the progression of HT-29 D4 cells from G1 to S phase. Northern blot analysis using a dipeptidyl peptidase i.v. probe revealed that the growth arrest mediated by IAF was not linked to differentiation processes. By Western blotting with polyclonal antibodies against IAF, we found that this protein was not detected in differentiated colonic carcinoma. Our results suggest that IAF might regulate intestinal cell proliferation.     

Purification and properties of an endothelial cell growth factor from human platelets

An endothelial cell growth factor has been purified about 1,000,000-fold to homogeneity from human platelets by a seven-step procedure. The purified product has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 45,000. The mobility in sodium dodecyl sulfate gel electrophoresis was similar in the presence or absence of reducing agents, indicating that the factor consists of a single polypeptide chain. Maximal stimulation by the purified protein was achieved at a concentration of about 20 ng/ml (440 pM). Heparin did not potentiate the activity, nor did the factor bind to heparin immobilized on Sepharose. The purified factor was heat- and acid-labile; it was active on porcine and human endothelial cells, but not on human foreskin fibroblasts. Chromatofocusing revealed that the pI of the factor was 4.6. The structural and functional characteristics of the platelet-derived endothelial cell growth factor are distinct from previously characterized endothelial cell mitogens with affinities for heparin.     

Purification and properties of trehalase from monkey small intestine

Brush border membrane trehalase was purified from monkey small intestine by a procedure which includes solubilisation by Triton X-100, ammonium sulphate fractionation, and chromatography on DE-52 and hydroxyapatite. The purified enzyme had a specific activity of 11 units/mg protein and was purified 140-fold. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis. It had aK ^m value of 17.4 mM for trehalose and a V_max of 1.33 units. Sucrose and Tris acted as competitive inhibitors of the enzyme.     

Purification and properties of an acid deoxyribonuclease from rat small intestinal mucosa

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.     

Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa

An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose. The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4. It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+. Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide. The enzyme does not attack RNA and is inhibited by it. Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000. The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation. The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis. The enzyme degrades denatured DNA about 20 times faster than native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini.     

Hydroxyxanthone as an inhibitor of cAMP-activated apical chloride channel in human intestinal epithelial cell

AimsPrevious investigation showed that polyphenols abundantly found in many plants could inhibit Cl^? secretion. The present study was aimed to investigate the effect of phenol containing xanthone derivatives on cAMP-activated intestinal Cl^? secretion and evaluate potential benefits of these compounds in the treatment of cholera.Main methodsFour hydroxy xanthones were synthesized via oxidative coupling reaction of the corresponding ortho-hydroxybenzoic acids and hydroxyphenols. Short-circuit current and apical Cl^? current measurements across monolayers of human intestinal epithelial (T84) cell and Fisher rat thyroid cells transfected with human CFTR (FRT-hCFTR cell) were performed to determine the effect of hydroxyxanthones on cAMP-activated Cl^? secretion. Intracellular cAMP was measured by immunoassay methods. Anti-diarrheal efficacy was evaluated using closed loop model of cholera.Key findingsAmong the tested xanthones, 1,3,6-trihydroxyxanthone (THX-001) was found to be the most potent derivative in the inhibition of cAMP-activated Cl^? secretion across T84 cell monolayers (IC₅₀ ~ 100 μM). Electrophysiological analysis of T84 cells and FRT-hCFTR cells revealed that THX-001 targeted two distinct cAMP-activated Cl^? channels in the apical membrane of T84 cells, namely, CFTR and inward rectifying Cl^? channel (IRC). In contrast, THX-001 had no effect on intracellular cAMP levels in these cells. Importantly, THX-001 completely abolished cholera toxin-induced Cl^? secretion across T84 cell monolayers and significantly inhibited cholera toxin-induced intestinal fluid secretion in mouse closed loop models.SignificanceThis study revealed that hydroxyxanthone represents another chemical class of polyphenolic compounds that may hold promise as anti-secretory therapy for cholera.     

Differentiation of intestinal epithelial cell line (IEC-18) by an acid extract of rat small intestine

A factor which may induce differentiation of intestinal epithelial cell lines in vitro was found in an acid extract of adult rat small intestine. The addition of a partially purified acetic acid extract of rat small intestine to IEC-18 cell culture dishes increased sucrase activity within 48 h. Thymidine incorporation markedly decreased within 24 h. Significant development of microvilli-like structures was observed on the acid extract-treated IEC-18 cells, compared with controls. This activity of rat acid extract was heat-stable and the apparent molecular weight of the factor was 400-800. These findings suggested that the factor may be related to the epithelial differentiation of rat small intestinal crypt cells.     

Purification and properties of epithelial growth inhibitor (EGI) from human platelets: its separation from type beta transforming growth factor (TGF-beta)

We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-ethanol extract of human platelets. During purification, EGI was separated from the major component of type beta transforming growth factor (TGF-beta), which can stimulate the colony formation of the non-malignant fibroblastic cell line derived from rat kidney (NRK) in soft agar in the presence of epidermal growth factor (EGF). The purified EGI had an Mr of 27,000, and was composed of two subunits identical in Mr. It significantly inhibited the growth in monolayer cultures of three non-malignant epithelial cell lines, BRL, MDCK (from Madin-Darby canine kidney) and BSC-1 (from African green monkey kidney), at doses lower than 40 pg/ml in medium containing 10% fetal calf serum. Its inhibitory activity was stable against heating at 90 degrees C for 3 min, but not against treatment with 50 mM dithiothreitol. In addition, TGF-beta was also partially purified from the same extract. The purified TGF-beta did not show any inhibitory activity toward the growth of BRL, MDCK, BSC-1, or NRK.     

Purification of a cell growth inhibitor from bovine cerebral cortex cells

A glycopeptide, isolated from bovine cerebral cortex cells and added in only nanogram levels to cells in culture, has been shown to inhibit both cell protein synthesis and cell division. When purified by gel filtration and $\text{Ulex}$$\text{europaeus}$ lectin affinity chromatography, the radioiodinated preparation was subjected to high resolution isoelectric focusing and shown to contain three species of macromolecules. The glycopeptide focusing at pH 8.1 comprised over 75% of the radioiodinated material and possessed inhibitory activity against both cell protein synthesis and cell division. A second species that focused at pH 8.3 was also found to be inhibitory to cell metabolism and may have represented a variant of the major glycopeptide.     

Purification and properties of a DNase inhibitor from Nicotiana tabacum cell cultures

Extraction of Nicotiana tabacum cell cultures, chromatography on DEAE-cellulose and gel filtration resulted in a homogeneous protein (Mr = 14500), which strongly reduces the hydrolysis of Escherichia coli DNA by DNase I. DNA degradation by micrococcal nuclease is not inhibited. The inhibitor protein interacts with DNase I in the absence of DNA, as determined by the partial quenching of protein intrinsic fluorescence; a 1:1 stoichiometry is deduced. From the reduction of DNase I activity with increasing inhibitor concentration apparent equilibrium constants for the inhibitor X DNase-I complex have been calculated. This interaction is strongly temperature-dependent; at 20 degrees C and 26 degrees C dissociation constants of 5 nM and 110 nM, respectively, were determined. As a consequence a rather high enthalpy of interaction can be estimated.     

Ribonuclease inhibitor from human placenta. Purification and properties

A soluble ribonuclease inhibitor from the human placenta has been purified 4000-fold by a combination of ion exchange and affinity chromatography. The inhibitor has been isolated in 45% yield (about 2 mg/placenta) as a protein that is homogeneous by sodium dodecyl sulfate-gel electrophoresis. In common with the inhibitors of pancreatic ribonuclease from other tissues that have been studied earlier, the placental inhibitor is an acidic protein of molecular weight near 50,000; it forms a 1:1 complex with bovine pancreatic RNase A and is a noncompetitive inhibitor of the pancreatic enzyme, with a Ki of 3 X 10(-10) M. The amino acid composition of the protein has been determined. The protein contains 30 half-cystine plus cysteine residues determined as cysteic acid after performic acid oxidation. At pH 8.6 the nondenatured protein alkylated with iodoacetic acid in the presence of free thiol has 8 free sulfhydryl groups. The inhibitor is irreversibly inactivated by sulfhydryl reagents and also by removal of free thiol from solutions of the protein. Inactivation by sulfhydryl reagents causes the dissociation of the RNase - inhibitor complex into active RNase and inactive inhibitor.     

Purification and properties of a subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus

A subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus were purified to homogeneity by conventional methods such as chromatography on DEAE-cellulose, gel filtration on Sephadex G-75, PAGE and affinity chromatography. The final preparations were homogeneous on PAGE. Their pI were 7.66 and 7.70, respectively. The dissociation constant of the complex of the inhibitor with subtilisin was 2.69.10(-10) M. Both the inhibitors were stable to heat, TCA and ethanol. The molecular weights of the subtilisin inhibitor and the associated trypsin inhibitor by gel filtration were 7500 and 8200, respectively.     

Purification and properties of alkaline phosphatase from the mucosa of rat small intestine

Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, EC 3.1.3.1] was purified from the mucosa of rat small intestine by butanol extraction, ethanol fractionation, gel filtration, with controlled-pore glass-10 and DEAE-cellulose column chromatography. On the gel filtration, the enzyme activity was separated into three peaks; A in the void volume, B and C at lower molecular weight positions. Enzyme A was purified to homogeneity. The activity of enzymes A, B, and C was detected even on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at the position of the protein of enzyme A, which had a molecular weight of 110,000 daltons. Enzymatic properties such as pH optimum, Km value for the substrate, heat inactivation and inhibition by amino acids were the same in all three enzymes. Based on these findings, together with the elution positions on gel filtration, enzyme A was regarded as an aggregate, and enzymes B and C as dimer and monomer molecules, respectively.     

Establishment of intestinal epithelial cell lines from adult mouse small and large intestinal crypts

Small and large intestinal epithelial cell (IEC) lines were established from adult murine intestinal crypts. Both established small and large IECs line (named aMoS7 and aMoC1 respectively) expressed epithelial markers. Similarly to IECs isolated from adult mouse intestines, the expression of major histocompatibility complex (MHC) class II molecules was induced by interferon-γ-treatment in both established cell lines. This expression of MHC class II molecules was higher in small intestinal aMoS7 cells than in large intestinal aMoC1 cells. Treatment with lipopolysaccharide and with ligands of Toll-like receptors 1, 2, 3, and 7 induced secretion of interleukin-6 from both adult IEC lines. These results suggest that the aMoS7 and aMoC1 cell lines can serve as useful tools in analyzing the immunological functions of IECs, especially in studying the IEC response to microbial components and its antigen presenting ability.     

Purification and properties of human intestine alanine aminopeptidase

Human intestinal alanine aminopeptidase has been purified to greater than 90% homogeneity. The enzyme was released from mucosal cell membranes by Triton X-100 treatment. The native enzyme had a molecular weight of 206,000 in dilute buffer and 108,000 in the presence of sodium dodecyl sulfate. The enzyme was inhibited by chelators suggesting the presence of a metal ion in the enzyme. The most potent chelator inhibitor tested, o-phenanthroline, gave mixed kinetics (Ki = 67 micro M). Activity was restored by removal of the chelator. The enzyme was inhibited competitively by amino acids having hydrophobic side chains such as L-phenylalanine (Ki = 0.67 mM). Puromycin and methicillin also inhibited the enzyme in the competitive (Ki = 12.5 micro M) and noncompetitive (Ki = 4.6 mM) manner, respectively. Kinetic analysis of several amino acid beta-naphthylamides as substrates demonstrated the preference for substrates having hydrophobic or basic amino terminal residues with no beta-branching. L-Methionyl-beta-naphthylamide was the most tightly bound with L-alanyl-beta-naphthylamide was the most rapidly hydrolyzed.     

Purification and properties of growth inhibitor from normal rabbit serum.

It was previously found that rabbit serum contains a growth-inhibitory substance for a tumorigenic rat liver cell line RSV-BRL. In the present study, the growth inhibitor was purified from normal rabbit serum to show a homogeneous protein band with a molecular weight (Mr) of 56 k on SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The purified growth inhibitor, tentatively named rabbit serum-derived growth inhibitor (RSGI), potently inhibited the growth of RSV-BRL and nine kinds of other cell lines including three human tumor cell lines at a concentration of 20 ng/ml or higher. The growth-inhibitory effect of RSGI was reversible and appeared to be cytostatic rather than cytotoxic. RSGI was stable to heating at 56 degrees C for 30 min or treatment with 0.1 M 2-mercaptoethanol, but labile to heating at 100 degrees C for 3 min or treatment with 1 M acetic acid (pH 2.3), 6 M urea, 50% (v/v) 1-propanol, or 0.1% (w/v) trypsin. These properties of RSGI suggested that it was different from type beta transforming growth factors, tumor necrosis factor-alpha, and other known growth-regulatory factors.     

Purification and properties of an alpha-amylase inhibitor from wheat.

Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.