Intestinal Absorption of Dipeptides Containing Glycine, Phenylalanine, Proline, β-Alanine or Histidine in the Rat

1973 ◽  
Vol 45 (6) ◽  
pp. 849-858 ◽  
Author(s):  
D. J. Boullin ◽  
R. F. Crampton ◽  
Christine E. Heading ◽  
D. Pelling
Keyword(s):  
Intestinal Absorption ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Peptide Absorption ◽  
Physiological Conditions ◽  
Anaesthetized Rats ◽  
Tissue Homogenates ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

1. The intestinal absorption of carnosine, glycylglycine, glycyl-d-phenylalanine, glycyl-l-phenylalanine, glycyl-l-proline and l-prolylglycine were investigated after intraluminal injection of dipeptide into anaesthetized rats. 2. With all six dipeptides, the intact substance was detected by ion-exchange chromatography in blood samples taken from the superior mesenteric vein. 3. The rate of hydrolysis of the dipeptides in tissue homogenates was measured in vitro. 4. The relative rates of hydrolysis varied by a factor of 300; there was an apparent inverse relationship between rate of hydrolysis and detection of intact peptide. 5. Peptide absorption was accompanied by increases in venous concentrations of the component amino acids, which appeared in proportions appropriate to the view that peptide absorption preceded hydrolysis. 6. It is suggested that slowly hydrolysed dipeptides may pass intact through the intestine wall under physiological conditions.

scholarly journals Liberation of tryptic fragments from caseinomacropeptide of bovine κ-casein involved in platelet function. Kinetic study

1990 ◽  
Vol 271 (1) ◽  
pp. 247-252 ◽  
Author(s):  
J Léonil ◽  
D Mollé
Keyword(s):  
Platelet Function ◽  
Acid Precipitation ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Rate Of Hydrolysis ◽  
The Difference ◽  
Cationic Exchange ◽  
Hydrolysis Of ◽  
Similar Kinetic

Carbohydrate-free caseinomacropeptide (CMP) was purified from rennet-hydrolysed caseinate by trichloroacetic acid precipitation and DEAE-TSK Fractogel-650 ion-exchange chromatography. To study the liberation of 106-112, 106-116 and 113-116 fragments from carbohydrate-free CMP involved in platelet function, a quantitative study was made on the rate of hydrolysis of the three peptidic bonds that are susceptible to the action of trypsin. Data were obtained from reverse-phase (Ultrabase column) and cationic-exchange (Mono S column) h.p.l.c. On the basis of the disappearance of substrate, kcat. and Km were respectively 3.95 s-1 and 0.2 mM. The two 111-112 and 112-113 bonds were split according to similar kinetic parameters (kcat. = 1.97 s-1, Km = 0.2 mM) and much faster than the 116-117 bond. The difference in susceptibility of the bonds can probably be attributed to the nature of residues flanking the primary proteolytic sites rather than to their accessibility to the proteinase. On the basis of our results the 106-116 fragment cannot be formed.

Degradation of endogenous heptadecapeptide gastrin by endopeptidase 24.11 in the pig

1987 ◽  
Vol 253 (1) ◽  
pp. G33-G39
Author(s):  
D. M. Power ◽  
N. Bunnett ◽  
A. J. Turner ◽  
R. Dimaline
Keyword(s):  
Specific Inhibitor ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Antral Mucosa ◽  
Venous Outflow ◽  
Endopeptidase 24.11 ◽  
Specific Antisera ◽  
Hydrolysis Of

Hydrolysis of heptadecapeptide gastrin (G-17) by endopeptidase 24.11 (EC 3.4.24.11) was studied in vivo and in vitro in the pig. Ion exchange chromatography and radioimmunoassay with three region-specific antisera were used to identify the products of porcine G-17 degradation. Incubation of antral extracts with pure endopeptidase 24.11 resulted in a substantial loss of intact G-17: 80% C-terminal immunoreactivity was lost in 60 min. This hydrolysis was completelyinhibited by phosphoramidon, which is a specific inhibitor of endopeptidase 24.11. In antral extracts G-17 accounted forgreater than 95% of total C-terminal immunoreactivity, compared with less than 60% C-terminal immunoreactivity in the gastric venous outflow; shorter C-terminal forms comprised the major part of the remaining immunoreactivity. After infusion of phosphoramidon, the concentration of intact G-17 was increased, and there was a corresponding reduction in the concentration of other C-terminal immunoreactive fragments. We conclude that endopeptidase 24.11 degrades G-17 in vitro and in vivo and may be responsible for the generation of C-terminal fragments from G-17 after secretion from the porcine antral mucosa.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

Repeated thyrotrophin stimulation of thyroid secretion: lack of refractoriness in vivo

1985 ◽  
Vol 106 (2) ◽  
pp. 153-157
Author(s):  
N. Bagchi ◽  
T. R. Brown
Keyword(s):  
Adenylate Cyclase ◽  
Thyroid Tissue ◽  
Control Group ◽  
Cyclic Amp Accumulation ◽  
Thyroid Glands ◽  
Rate Of Hydrolysis ◽  
Thyroid Secretion ◽  
Hydrolysis Of

ABSTRACT It has been reported that prior exposure of thyroid tissue to TSH in vitro induces a state of refractoriness to new challenges of the hormone. We have investigated the effect of repeated TSH treatment on thyroid secretion to determine whether such refractoriness exists in vivo. The rate of thyroid secretion was estimated by measuring the rate of hydrolysis of labelled thyroglobulin from mouse thyroid glands in vitro. The thyroid glands were labelled in vivo with 131I and then cultured for 20 h in the presence of mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the percentage of radioactivity released as free iodotyrosines and iodothyronines into the gland and the medium at the end of incubation. Thyrotrophin was administered in vivo at hourly intervals for 2–4 injections. The corresponding control group received saline injections every hour except for the last injection when they received TSH. The peak rates of thyroglobulin hydrolysis, measured 2 h following the last injection, were similar in animals receiving two, three or four TSH injections and were not different from those in the control groups. Serum tri-iodothyronine and thyroxine concentrations 2 h after the last injection were higher in the groups receiving multiple TSH injections. Thyroidal cyclic AMP accumulation in response to TSH was markedly depressed in the group receiving multiple injections compared with the group receiving a single injection of TSH in vivo. These data indicate that (1) the stimulatory effect of TSH on thyroidal secretion is not diminished by prior administration of the hormone in vivo, (2) repeated TSH administrations in vivo cause refractoriness of the adenylate cyclase response to TSH and (3) a dichotomy exists between the secretory response and the adenylate cyclase response to repeated administrations of TSH. J. Endocr. (1985) 106, 153–157

scholarly journals Anionic amino acids support hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

2020 ◽  
pp. jbc.RA120.015524
Author(s):  
Alexandra P Breslawec ◽  
Shaochi Wang ◽  
Crystal Li ◽  
Myles B Poulin
Keyword(s):  
Amino Acids ◽  
Bacterial Infections ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Binding Surface ◽  
Activity Measurements ◽  
Rate Of Hydrolysis ◽  
Biofilm Dispersal ◽  
Hydrolysis Of

The exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm dependent bacterial infections. The cationic charge resulting from partial de-N-acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de-N-acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the –1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

scholarly journals Hydrolysis of14C-labelled proteins by rumen micro-organisms and by proteolytic enzymes prepared from rumen bacteria

1983 ◽  
Vol 50 (2) ◽  
pp. 345-355 ◽  
Author(s):  
R. J. Wallace
Keyword(s):  
Proteolytic Enzymes ◽  
Rumen Fluid ◽  
Performic Acid ◽  
Cross Linking ◽  
Acid Oxidation ◽  
Rumen Bacteria ◽  
Rate Of Hydrolysis ◽  
Micro Organisms ◽  
Hydrolysis Of

1. Proteins were labelled with14C in a limited reductive methylation using [14C]formaldehyde and sodium borohydride.2. The rate of hydrolysis of purified proteins was little (< 10%) affected by methylation and the14C-labelled digestion products were not incorporated into microbial protein during a 5 h incubation with rumen fluid in vitro. It was therefore concluded that proteins labelled with14C in this way are valid substrates for study with rumen micro-organisms.3. The patterns of digestion of14C-labelled fish meal, linseed meal and groundnut-protein meal by rumen micro-organisms in vitro were similar to those found in vivo.4. The rates of hydrolysis of a number of14C-labelled proteins, including glycoprotein II and lectin from kidney beans (Phaseolus vulgaris), were determined with mixed rumen micro-organisms and with proteases extracted from rumen bacteria. Different soluble proteins were digested at quite different rates, with casein being most readily hydrolysed.5. Proteins modified by performic acid oxidation, by cross-linking using 1,6-di-iso-cyanatohexane or by diazotization were labelled with14C. Performic acid treatment generally increased the susceptibility of proteins to digestion, so that the rates of hydrolysis of performic acid-treated proteins were more comparable than those of the unmodified proteins. Cross-linking resulted in a decreased rate of hydrolysis except with the insoluble proteins, hide powder azure and elastin congo red. Diazotization had little effect on the rate of hydrolysis of lactoglobulin and albumin, but inhibited casein hydrolysis and stimulated the breakdown of γ-globulin.

False-negative results for urinary phenothiazines and imipramine in Forrest's qualitative assays.

1980 ◽  
Vol 26 (2) ◽  
pp. 345-347 ◽  
Author(s):  
G P James ◽  
M H DJang ◽  
H H Hamilton
Keyword(s):  
Ascorbic Acid ◽  
Ion Exchange ◽  
Exchange Resin ◽  
Anion Exchange Resin ◽  
False Negative ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Negative Results ◽  
False Negative Results

Abstract When a series of patients' urine samples supplemented in vitro with chlorpromazine or imipramine was assayed with the Forrest qualitative assays, we observed an occasional false-negative result, which we found was attributable to interference by ascorbic acid. It interferes with the reagent, not with the analytes, in both assays. We easily eliminated this interference with the phenothiazine test by using an anion-exchange resin. Eliminating the interference with the assay for imipramine, however, is more difficult; false-negative results can be obtained even after ion-exchange chromatography if the imipramine concentration is less than 50 mg/L.

Effect of 2,4-D on the Hydrolysis of Diclofop-Methyl in Wild Oat (Avena fatua)

Weed Science ◽  
1980 ◽  
Vol 28 (6) ◽  
pp. 725-729 ◽  
Author(s):  
B. D. Hill ◽  
B. G. Todd ◽  
E. H. Stobbe
Keyword(s):  
Enzyme Preparation ◽  
Avena Fatua ◽  
Wild Oat ◽  
Enzymic Hydrolysis ◽  
Standard Assay ◽  
Rate Of Hydrolysis ◽  
Dichlorophenoxy Acetic Acid ◽  
Hydrolysis Of ◽  
Assay Conditions

The basis for 2,4-D [(2,4-dichlorophenoxy)acetic acid] antagonism of diclofop-methyl {methyl 2-[4-(2,4-dichlorophenoxy) phenoxy] propanoate} toxicity to wild oat (Avena fatuaL.) was investigated by studying changes in the metabolism of diclofop-methyl in vitro. An esterase from wild oat, which hydrolyzes diclofop-methyl to the acid diclofop, was extracted, partially purified, and the reaction characterized. The rate of hydrolysis of14C-diclofop-methyl was 0.14 ηmoles/2 h at standard assay conditions of 0.25 mg lyophilized enzyme preparation (19.6% protein) in 0.1 ml phosphate buffer (0.1 M, pH 7.0), substrate 5 μM. The addition of 2,4-D to this reaction did not inhibit14C-diclofop formation. Higher levels of 2,4-D stimulated enzymic hydrolysis.14C-diclofop-methyl was rapidly metabolized to14C-diclofop and polar14C-conjugates when vacuum-infiltrated into wild oat leaf segments. The addition of 2,4-D caused small increases in the rates of both14C-diclofop-methyl de-esterification and14C-diclofop conjugation. It is concluded that 2,4-D does not inhibit the in vitro de-esterification of diclofop-methyl.

Automated analysis for taurine in biological fluids and tissues.

1987 ◽  
Vol 33 (6) ◽  
pp. 835-837 ◽  
Author(s):  
H O Goodman ◽  
Z K Shihabi
Keyword(s):  
Ion Exchange ◽  
Continuous Flow ◽  
Biological Fluids ◽  
Automated Analysis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fluorescent Compound ◽  
Automated Method ◽  
Tissue Homogenates ◽  
Proteins And Peptides

Abstract We have developed an automated method of analysis for taurine, based on incorporating an ion-exchange chromatography column into the continuous-flow AutoAnalyzer (Technicon). After removal of proteins and peptides by dialysis, taurine is selectively eluted from an ion-exchange column and reacted with o-phthaldialdehyde to yield a fluorescent compound. The advantages of this method are: full automation with no need for sample deproteinization or cleanup; sensitivity, detecting as little as 5 mumol/L; speed (20 samples per hour); and flexibility. It can be used for assaying taurine in urine, plasma, cerebrospinal fluid, and tissue homogenates. This method can be adapted for assays of other metabolites.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates

2009 ◽  
Vol 55 (7) ◽  
pp. 874-878 ◽  
Author(s):  
Vijay Prabha ◽  
Tanushree Gupta ◽  
Siftjit Kaur ◽  
Navchetan Kaur ◽  
Sushila Kala ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Spermatozoa ◽  
Infertile Woman ◽  
Ammonium Sulfate Precipitation ◽  
Tail Region ◽  
Sperm Immobilization

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 °C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an ~20 kDa protein. SIF at a concentration of 10 µg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 °C, whereas a concentration of 150 µg/mL caused immediate immobilization, and a concentration of 200 µg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin–nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.

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