Effect of bovine small intestine thioredoxin on aldose reductase activity

Under oxidative stress mediated by H₂O₂, significant activation of purified aldose reductase from bovine small intestine was observed in the presence of purified thioredoxin from bovine small intestine.     

Inhibitory effects of Ge-132 (carboxyethyl germanium sesquioxide) derivatives on enkephalin-degrading enzymes

Twenty-eight species of carboxyethyl germanium sesquioxide (Ge-132) derivatives were examined for their inhibitory effects on enkephalin-degrading enzymes that were purified from monkey brain, the longitudinal muscle layer of bovine small intestine, and human cerebrospinal fluid (CSF). A series of the sulfurized Ge-132 derivatives showed strong inhibition of these purified enzymes. The most effective ones were Ge-014 and Ge-107, which showed IC50 values of 60 and 100 micrograms/ml, respectively, for dipeptidylcarboxypeptidase from the longitudinal muscle layer. They also exhibited inhibitory activity against aminopeptidase from human CSF, the IC50 values being 450 and 440 micrograms/ml, respectively. Furthermore, these compounds showed inhibition of dipeptidylaminopeptidase from monkey brain and the longitudinal muscle layer of bovine small intestine. These compounds are expected to have analgesic effects due to their inhibition of the degradation of endogenous opioid peptides.     

Influence of thyrocalcitonin on the periodic motor activity of the gastro-intestinal tract]

Experiments were conducted on dogs with fistulae of the stomach and various portions of the small intestine in which there was recorded a periodic motor activity. It was shown that intravenous injection of a highly purified bovine thyrocalcitonin (TCT) in doses of 2--10 Units/kg (considerably exceeding the doses used at the clinic in the treatment of patients with bone pathology) caused no changes in a number of indices characterizing the motor periodic activity of the stomach and various portions of the small intestine, despite a distinct reduction (by 10--20%) of the calcium level in the blood serum.     

Molecular, biochemical and immunological analyses of porcine pancreatic DNase I.

Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.     

Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): sequence characterisation, expression pattern and characterisation of proteolytic activity

Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51-58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2.     

Lithocholate binding by Y and Y' proteins in bovine small intestine.

Cytosolic proteins may play an important role in the intracellular transport of bile acids in enterocytes. The lithocholate binding properties of cytosolic protein from bovine small intestine were studied. Lithocholate binding was observed in the Y (45-50 kDa), Y' (30-35 kDa), and Z fractions (10-15 kDa) following gel filtration of cytosol. A Y protein with glutathione S-transferase activity (46 kDa) was purified by S-octyl-glutathione affinity chromatography and chromatofocusing (eluted at pH 7.5) of the Y fraction. Two Y' bile acid binding proteins with dihydrodiol dehydrogenase activity were partially purified from the Y' fraction by chromatofocusing and hydroxyapatite-HPLC. The lithocholate binding affinity of Y' protein (Kd < 0.35 microM) was higher than that of Y protein (Kd = 2 microM) and was comparable to that of Z protein (Kd = 0.2 microM). The binding affinity of Y protein was higher for bilirubin (Kd = 2.5 microM) than that for BSP (Kd = 200 microM). This was comparable to the binding affinity of bovine hepatic Y protein. These data indicate that Y' and Z proteins participate in the intracellular transport of bile acids from the brush border to the basolateral pole in enterocytes.     

Purification and amino acid sequence of vasoactive intestinal peptide, peptide histidine isoleucinamide and secretin from the ovine small intestine

Vasoactive intestinal peptide (VIP), peptide histidine isoleucinamide (PHI) and secretin were separated and purified to homogeneity from ovine small intestine, using radioimmunoassay and radioreceptor assay for detection. An efficient and rapid purification sequence included acid extraction, concentration on a bulk C18 cartridge, filtration on a Fractogel column, ion-exchange chromatography on Mono-S and a maximum of three successive reverse-phase HPLC steps. The amounts of peptides obtained from 450 g wet weight tissue were 20 micrograms VIP, 15 micrograms PHI and 5 micrograms secretin. The as yet unknown amino acid sequences of the three peptides were found to be identical to those of the corresponding bovine peptides.     

Xanthine oxidase activity and immunologically detectable protein in the C57B1/6 mouse

1. Xanthine oxidase (XO) was purified from livers of C57B1/6 mice. Antibodies generated against the purified protein were used in an immunoassay to measure total XO protein. 2. Both the specific activity and amount of XO protein were greater in the proximal small intestine than in the liver. A pool of inactive enzyme was present in the small intestine which developed after weaning. 3. Male C57B1/6 mice had the same XO specific activity as females and neither the hepatic nor the intestinal XO activity were affected by the level of dietary protein. 4. When mice were fed diets with tungsten, XO activity was lost and the amount of XO protein in the small intestine was decreased.     

Tryptophan 5-hydroxylase in rat intestine

Tryptophan 5-hydroxylase was partially purified from rat small intestine and characterized. The enzyme activity was mainly localized in the distal one-fourth of the small intestine. The enzyme required Fe(2+), 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine and oxygen for full activity. The pH optimum of the reaction was 8.0. The hydroxylation rate of d-tryptophan by the enzyme was one-third that of l-tryptophan. l-Phenylalanine and l-tyrosine could not serve as substrates. The physiological significance of the enzyme is discussed.     

Studies on new intracellular proteases in various organs of rat. 1. Purification and comparison of their properties.

1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.     

Isolation,structure and biologic activity of chicken intestinal neurotensin

Using a radioimmunoassay towards bovine neurotensin (NT), chicken NT has been purified to homogeneity from extracts of intestine and its amino acid sequence determined to be: <Glu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu-OH. The molecule is identical to the bovine peptide except for the 3 amino acid substitutions located in its NH₂-terminal half and italicized above (His/Tyr; Val/Glu; Ala/Pro). The structure for chicken NT is consistent with earlier immunochemical studies which indicated a COOH-terminal homology with bovine NT [1]. The peptide isolated was shown to be near equipotent with bovine NT in its ability to induce hypotension, hyperglycemia, and cyanosis in the anesthesized rat, underscoring the importance of the COOH-terminal residues in NT for biological activity.     

Effect of concentrate feeding on the bovine intestinal and pancreatic carbohydrases. Evidence of induced increase in their activities

1. The effects of concentrate feeding on the levels and pattern of distribution of carbohydrases in bovine intestine and pancreas were investigated. 2. No remarkable difference was noticed in the pattern of distribution of the carbohydrases along the bovine intestine, which was mostly confined to the proximal part of the small intestine. 3. The concentrate feeding, however, highly affected the levels of carbohydrases in the mucosa, luminal contents and the pancreas. Their levels slightly decreased in the mucosal tissue and significantly increased in the luminal contents. In the pancreas, the level of amylase decreased and that of disaccharidases increased. 4. Based on the presence of higher levels of activities of carbohydrases in the luminal contents, supported by the concentrate-induced increase in their levels, it is argued that the site of carbohydrate digestion, including disaccharides, in the small intestine, is the luminal contents.     

Studies on the formation of gamma-aminobutyric acid from putrescine in rat organs and purification of its synthetic enzyme from rat intestine.

The formation of gamma-aminobutyric acid (GABA) from putrescine was examined in organs of rats using radioactive putrescine. Radioactive GABA was detected in all the organs of a rat injected intraperitoneally with radioactive putrescine, and the highest radioactivity of GABA was observed in the small intestine. The enzyme involved in this formation was purified from small intestine and identified as a diamine oxidase, histaminase, from the properties of the enzyme. Activity of the enzyme was found in all the organs of rat, and the highest activity was observed in the small intestine.     

Involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing intestinal IEC-6 cell death

Monomeric 14-kDa bovine alpha-lactalbumin was purified with a preparation of lower molecular weight whey protein concentrate from Holstein cow normal milk followed by size exclusion chromatography. The protein showed a stimulatory rather than an inhibitory effect on the proliferation of a cultured IEC-6 cell line from the rat small intestine. But incubation in 30% trifluoroethanol/acetate buffer (pH 5.5) at 37 degrees C for 5 d in a slowly rotating test tube rendered it highly cytotoxic with concomitant appearance of SDS-stable 20- and 30-kDa forms of alpha-lactalbumin on electrophoresis. Furthermore, alpha-lactalbumin obtained by a one-step purification procedure by affinity chromatography on an anti-alpha-lactalbumin antibody column from the lower molecular weight whey protein concentrate, which had been found to contain several SDS-stable higher M(r) forms of alpha-lactalbumin, exhibited potent inhibitory activity on IEC-6 cell growth. These results indicate the involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing cell death on the intestinal IEC-6 cell line.     

Prolidase from bovine intestine: purification and characterization

Prolidase [iminodipeptidase, EC 3.4.13.9] was highly purified from the cytosol fraction of bovine small intestine by a series of column chromatographies on DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose and hydroxyapatite. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.2 with Gly-Pro as substrate. It was stable between pH 5.5 and 8.5 for 30 min at 30 degrees C and retained half of the activity after 15 min at 40 degrees C. It was completely inactivated by p-chloromercuribenzoate (PCMB) but not inhibited by diisopropylphosphorofluoridate (DFP), phenylmethane sulfonylfluoride (PMSF) and metal chelators. Its amino acid composition was determined. Its molecular weight was estimated to be 116,000 by gel filtration on Sephadex G-150 and 56,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that it is a dimer. It hydrolyzed dipeptides represented as X-Pro (X = amino acid).     

Purification and characteristics of functional properties of soluble nucleoside triphosphatase (apyrase) from bovine brain

Soluble NTPase, differing in its properties from known proteins exhibiting NTPase activity, was purified from bovine brain to homogeneity. The enzyme has pH optimum at 7.5 and shows absolute dependence on bivalent cations and broad substrate specificity towards nucleoside-5 -tri- and -diphosphates, characteristics of apyrases. The NTPase follows Michaelis-Menten kinetics in the range of investigated substrate concentrations, the apparent K(m) values for UTP, ITP, GTP, CTP, CDP, and ATP being 86, 25, 41, 150, 500, and 260 microM, respectively. According to gel-filtration and SDS-PAGE data, the molecular mass of the enzyme is 60 kD. The NTPase is localized in the cytosol fraction and expressed in different bovine organs and tissues. Total NTPase activity of extracts of bovine organs and tissues decreases in the following order: liver > heart > skeletal muscle > lung > brain > spleen > kidney ~ small intestine. The enzyme activity can be regulated by acetyl-CoA, alpha-ketoglutarate, and fructose-1,6-diphosphate acting as activators in physiological concentrations, whereas propionate exhibits an inhibitory effect.     

Isolation and characterization of MUC15, a novel cell membrane-associated mucin.

The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino-acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full-length cDNA encoding a protein of 330 amino-acid residues. N-terminal amino-acid sequencing of derived peptides and the purified protein confirmed 76% of the sequence and demonstrated presence of a cleavable signal peptide of 23 residues, leaving a mature protein of 307 amino acids. Database searches showed no homology to any other proteins. A survey of the human genome indicated the presence of a corresponding gene on chromosome band 11p14.3. Isolation and sequencing of the complete cDNA sequence of the human homologue proved the existence of the gene product (334 amino-acid residues). This novel mucin-like protein was named MUC15 by appointment of the HUGO Gene Nomenclature Committee. The deduced amino-acid sequences of human and bovine MUC15 demonstrated structural hallmarks characteristic for other membrane-bound mucins, such as a serine, threonine, and proline-rich extracellular region with several potential glycosylation sites, a putative transmembrane domain, and a short cytoplasmic C-terminal. We have shown the presence of O-glycosylations, identified N-glycosylations at 11 of 15 potential sites in bovine MUC15, and a splice variant encoding a short secreted mucin. Finally, analysis of human and bovine cDNA panels and libraries showed MUC15 gene expression in adult human spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone marrow, lymph node, tonsil, breast, fetal liver, bovine lymph nodes and lungs of both species.     

Quantitative mRNA analysis of eight bovine 5-HT receptor subtypes in brain, abomasum, and intestine by real-time RT-PCR

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5-hydroxytryptamine receptor (5-HTR)] expression were available so far. This study aimed to develop real-time RT-PCR assays for quantitative mRNA analysis of bovine 5-HTR subtypes. Because the bovine 5-HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real-time RT-PCR assays (partial CDS) for the following bovine 5-HTR subtypes were developed and validated: 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, and 5-HTR4. Intra- and inter-assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5-HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5-HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde-phosphate-dehydrogenase (GAPDH). The 5-HTR subtype expression levels ranged from 0.001% (5-HTR2C in intestine) to 1% 5-HTR/GAPDH (5-HTR1B and 5-HTR4 in intestine). There were high variations of 5-HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real-time RT-PCR assays for quantitative analysis of bovine 5-HTR subtype gene expression were developed and validated.     

Inhibition by palmitoyl CoA of dynein ATPase from sea urchin spermatozoa

ATPase of 14S dynein, extracted from spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, and partially purified by sucrose density gradient centrifugation, was inhibited non-competitively by palmitoyl CoA at concentrations higher than 20 microns, and was stimulated at concentrations between 2 microns and 10 microns. The effects of palmitoyl CoA on dynein ATPase were reversed by bovine serum albumin (1 mg/ml) and spermine (0.1 and 1 mM). Myristoyl CoA exerted effects similar to those of palmitoyl CoA. Short chain fatty acyl CoAs, such as butyryl CoA, propionyl CoA and acetyl CoA, CoA, Na-palmitate, Na-myristate, and palmitoyl carnitine had hardly any effect on dynein ATPase. Palmitoyl CoA failed to inhibit purified CF1 ATPase from chloroplasts of spinach, ATPase of rat liver mitochondria and alkaline phosphatase from calf intestine.