Calf Intestinal Mucosa Research Articles

Adsorption induced enzyme denaturation: The role of protein surface in adsorption induced protein denaturation on allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymers

The effects of protein size on adsorption and adsorption-induced denaturation of proteins on copolymers of allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) have been studied. Different responses were observed for the amount of protein adsorbed and denatured on the polymer surface for different proteins (trypsin, alchol dehydrogenase from baker's yeast (YADH), glucose dehydrogenase (GDH) from Gluconobacter cerinus, and alkaline phosphates from calf intestinal mucosa (CIAP). Protein adsorption on the copolymer with 25% crosslink density (AGE-25) was dependent not only on the size of the protein but also on the presence of glycoside residues on the protein surface. Adsorption and denaturation of proteins follows the order YADH > trypsin > GDH ≫ CIAP although the molecular weights of the proteins follow the order YADH > CIAP > GDH > trypsin. The lack of correlation between amount of adsorbed protein and its molecular weight was due to the presence of glycoside residues on CIAP and GDH which protect the enzyme surface from denaturation. Enzyme stabilities in aqueous solutions of 1-cyclohexyl-2-pyrrolidinone (CHP) correlate well with the trend in denaturation by the copolymer, strongly suggesting that hydrophobic interactions play a major role in protein binding and the mechanism of protein denaturation is similar to that for water–miscible organic solvents.

Interactions of Typical and Atypical EnteropathogenicEscherichia coliStrains with the Calf Intestinal Mucosa Ex Vivo

Enteropathogenic Escherichia coli (EPEC) can be found in healthy and diarrheic cattle; however, little is known about the role of attaching and effacing (A/E) lesion formation in colonization of bovine intestinal mucosa by such strains. We show that typical and atypical EPEC induce A/E lesions on calf intestinal explants independently of Tir tyrosine phosphorylation and TccP. Our data support the existence of conserved Tir- and TccP-independent mechanisms of A/E lesion formation in a range of hosts and reinforce the zoonotic potential of EPEC in cattle.

Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase.

Polyclonal antibodies (PAb) prepared against bovine milk alkaline phosphatase (ALP) were used to develop a competitive indirect (CI) ELISA. Anti-ALP PAb were specific for milk ALP and did not react with ALP from E. coli or bovine and calf intestinal mucosa. Anti-ALP PAb were 20% cross-reactive with bovine placenta ALP. The anti-ALP antibodies also did not recognize bovine serum albumin, acid glycoprotein, ovalbumin, ferritin, and casein, although some cross-reactivity was observed with whey protein isolate. Anti-ALP PAbs reacted with deglycosylated native ALP, but did not recognize ALP denatured at 100 degrees C in 2% SDS or deglycosylated denatured ALP. When buffered solutions of milk ALP were heated at 70 degrees C, ALP activity decreased at a faster rate than ALP content determined by CI-ELISA. The ELISA differentiated between native and heat denatured ALP. Further studies are warranted to determine if an ELISA can be used to verify pasteurization of fluid milk.

Enantiomeric syntheses of conformationally restricted D- and L-2', 3'-dideoxy-2',3'-endo-methylene nucleosides from carbohydrate chiral templates.

D- and L-2',3'-dideoxy-2',3'-endo-methylene nucleosides were synthesized as potential antiviral agents. The key intermediates 5-O-tert-butyldiphenylsilyl-D- and L-2,3-dideoxy-2, 3-endo-methylenepentofuranoses (20 and 33, respectively) were obtained by selective protection of the D- and L-2,3-dideoxy-2, 3-endo-methylenepentose derivatives 19 and 32 which were prepared from 1,2:5,6-di-O-isopropylidene-D-mannitol and L-gulonic gamma-lactone, respectively, and converted to 5-O-tert-butyldiphenylsilyl-D- and L-2,3-dideoxy-2, 3-endo-methylenepentofuranosyl acetates (21 and 34, respectively) or the chlorides 22 and 35. The acetates and chlorides were condensed with pyrimidine and purine bases by Vorbrüggen conditions or S(N)2-type condensation. Vorbrüggen conditions using the acetates gave mostly alpha-isomers. In contrast, S(N)2-type condensation using the chlorides greatly improved the beta/alpha ratio. From the synthesis, several D- and L-2',3'-dideoxy-2',3'-endo-methylene nucleoside analogues have been obtained, and their structures have been elucidated by NMR spectroscopy and X-ray crystallography. The synthesized D- and L-adenine derivatives were tested as substrates of adenosine deaminase, which indicated that the D-adenosine derivative 4a was a good substrate of a mammalian adenosine deaminase from calf intestinal mucosa (EC 3.5.4.4) while its L-enantiomer 10a was a poor substrate. Either the D-adenine derivative 4a or its L-enantiomer 10a did not serve as an inhibitor of the enzyme.

Milk Alkaline Phosphatase Purification and Production of Polyclonal Antibodies

ABSTRACTPurification was by electroelution from native polyacrylamide gels or by sequential use of three columns. Electroelution was faster and resulted in a higher yield (23.4 vs 1.6%) than column purification. The enzyme had a molecular mass of 187 kDa, and the isoelectric point ranged from 5.4 to 6.0. ALP purified by electroelution was used as the antigen to immunize rabbits for polyclonal antibody (PAb) production. Western blot analysis showed that PAbs cross‐reacted with bovine milk and placenta ALP, but did not cross‐react with ALP from calf or bovine intestinal mucosa, Escherichia coli or with other milk proteins.

On the validity of continuous spectrophotometric assays for adenosine deaminase activity: A critical reappraisal

Kinetic investigations on adenosine deaminase from calf intestinal mucosa by spectrophotometric monitoring of the reaction at 264, 270, or 228 nm show that this method does not produce artifactual inhibition by substrate excess up to 0.7 m m concentration, when either adenosine or 2′-deoxyadenosine are employed with calf adenosine deaminase. The evaluation of kinetic parameters for this system was carried out both by initial rate measurements and by numerical differentiation of time progress curves according to a recently published method ( S. C. Koerber and A. L. Fink, 1987, Anal. Biochem. 165, 75–87). The following results were obtained by the latter method at pH 7.0 and 30°C: for the conversion of adenosine to inosine, k cat = 251 ± 15 s −1, K Ms = 29.7 ± 2.8 μ m, K Mp = 613 ± 62 μ m; for the conversion of 2′-deoxyadenosine to 2′-deoxyinosine, k cat = 283 ± 17 s −1, K Ms = 22.4 ± 2.2 μ m, K Mp = 331 ± 35 μ m. At 285 nm, a slight negative deviation from Beer's law was observed for adenosine at concentrations higher than 0.9 m m. No deviation was found for inosine up to 2.0 m m at the same wavelenth.

Regulation of the catalytic activity and oligomeric composition of enzymes in reversed micelles of surfactants in organic solvents

The Phenomenon of the regulation of the catalytic activity of enzymes via changing their oligomeric composition in the system of reversed micelles of sodium bis(2-ethylhexy)sulfosuccinate (AOT) in octane was studied using z-chymotrypsin (CT) from bovine brain and alkaline phosphatase (AP) from calf intestinal mucosa. The dependences of the enzyme catalytic activity on the AOT hydration degree ( W=[H 2O][AOT]), the parameter determining the radius ( r 4) of the inner cavity of micelles, usually represent the bell-shaped curves. The maximal catalytic activity is observed at such W o when r 4 is equal to the size of the enzyme molecule. The position of this maximum strictly correlates with the enzyme oligomeric composition. Thus, in the case of CT this is observed at W o =12 when r 1 is equal to the radius ( r p ) of the CT globule. In the case of artificially produced conjugate containing six cross-linked CT molecules, this is observed at W o=43 when r 4 is equal to the radius of the sphere surrounding the absolute octahedron composed of six CT globules. The dependence of the catalytic activity of AP on Wo represent a curve with two maxima that are observed when r 4 is equal to r p of either AP monomer ( W=17) or AP dimer ( W o=25), Ultracentrifugation experiments revealed that variation of W o causes a change in the oligomeric composition of AP its transition from monomeric ( W o<20) to demeric form ( W o > 20). Hence, the observed maxima correspond to functioning of different oligomeric forms of AP.

Dephosporylation with Alkaline Phosphatase of Histone and Fibrinogen Phosphorylated with Protein Kinase Cin vitro

Alkaline phosphatase from calf intestinal mucosa dephosphorylated histone H1 and fibrinogen that had been phosphorylated with protein kinase C. The reaction velocity was dependent on the ionic strength of the buffer; decreasing with increasing concentration. The pH optimum was around 7, which is lower than pH-optima described for other kinds of substrates. (32P) phosphorylated fibrinogen was dephosphorylated about 20 times faster than (32P)phosphohistone on a weight basis and the reaction continued linearily with time for the longest time tested (3 hs) even at 37 degrees C. As alkaline phosphatase is present in the blood the possible physiological significance of the dephosphorylation of phosphofibrinogen is discussed.

Laser photobleaching leads to a fluorescence grade adenosine deaminase

The enzyme adenosine deaminase (adenosine amino-hydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at λ max ex = 350 nm and λ max em = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence ( λ max ex = 305 nm, λ max em = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

Affinity difference of adenosine deaminases for the purine riboside-epoxyactivated Sepharose 6B column

1. 1. The small-size adenosine deaminases ( M r = 35,000 and 43,000 ) in calf intestinal mucosa, frog liver and scallop adductor muscle and the large-size deaminase ( M r = 100,000 ) in frog liver probably formed by adding a conversion protein to the small enzyme, were tightly bound to the purine riboside affinity column. 2. 2. Binding of the other large-size enzymes ( M r = 140,000 ) in the midgut gland of scallop and mussel to the column was not successful. 3. 3. It seems that the binding difference does not depend on a change in affinity between active site and ligand but rather on the stereospecific positioning of active site in the enzyme molecules.

Metabolism of different molecular forms of adenosine deaminase intravenously infused into the rabbit

High- M r and monomeric adenosine deaminase were injected intravenously into the rabbit. The rates of clearance and sites of uptake of the enzymes were compared. Calf intestinal mucosa served as the source of monomeric adenosine deaminase. High- M r enzymes were assembled in vitro from the calf enzyme and adenosine deaminase-complexing proteins isolated from rabbit plasma or kidney. An immunoassay specific for calf adenosine deaminase was used to determine which organs took up the injected enzyme. The enzymes were cleared from circulation in the following order: monomeric adenosine deaminase > high- M r enzyme prepared with kidney complexing protein > high- M r enzyme prepared with plasma complexing protein. High- M r enzyme assembled with kidney complexing protein was taken up primarily by the liver. Complexing protein and adenosine deaminating activity were cleared from circulation at similar rates. This and other evidence indicate that kidney complexing protein and calf adenosine deaminase are taken up as a unit by the liver. In contrast, adenosine-deaminating activity was cleared more quickly from circulation than complexing protein in rabbits injected with enzyme prepared with plasma complexing protein. Immunoassay results indicated that calf adenosine deaminase was taken up principally by the kidney cortex and liver. Kidney was the major site of uptake of monomeric adenosine deaminase. Indirect immunoperoxidase staining was used to localize the calf enzyme in glomeruli and proximal renal tubules, the same areas in which rabbit complexing protein is localized. These results support the hypothesis that complexing proteins play a role in the clearance of adenosine deaminase from plasma [ W. P. Schrader and P. J. Bryer (1982) Arch. Biochem. Biophys. 215, 107–115 ].

Adenosine deaminase inhibitors. Synthesis and biological evaluation of (+/-)-3,6,7,8-tetrahydro-3-[(2-hydroxyethoxy)methyl]imidazo[4,5-d] [1,3]diazepin-8-ol and some selected C-5 homologues of pentostatin.

The synthesis of several analogues of (8R)-3-(2-deoxy-beta-D-erythro- pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol (pentostatin, 1a) is described. Ring closure of 2-amino-1-(5-amino-1H-imidazol-4-yl)ethanone dihydrochloride (3) with triethyl orthoacetate or triethyl orthopropionate gave the C-5 methyl and ethyl ketoaglycons, 6,7-dihydro-5-methylimidazo[4,5-d][1,3]diazepin-8(3H)-one (4b) and 5-ethyl-6,7-dihydroimidazo[4,5-d][1,3]diazepin-8(3H)-one (4c), respectively. Stannic chloride catalyzed condensation of the pertrimethylsilyl derivatives of 4b and 4c with a protected glycosyl halide afforded anomeric mixtures of ketonucleosides 3-(2-deoxy-3,5-di-O-p-toluoyl-beta- and -alpha-D-erythro-pentofuranosyl)-6,7-dihydro-5-methylimidazo[4,5-d] [1,3]diazepin-8(3H)-one (5b and 6b) and 3-(2-deoxy-3,5-di-O-p-toluoyl)-beta- and -alpha-D-erythro-pentofuranosyl)-5-ethyl-6,7-dihydroimidazo[4,5-d]- [1,3]diazepin-8(3H)-one (5c and 6c), respectively. Subsequent separation of the anomers, followed by deprotection and reduction of 5b, 6b, and 5c, afforded the respective 8R and 8S isomers. Stannic chloride catalyzed condensation of pertrimethylsilyl ketoaglycon 4a with 2-(chloromethoxy)-1-(p-toluoyloxy) ethane to give ketonucleoside 6,7-dihydro-3-[[2-(p-toluoyloxy)ethoxy] methyl]imidazo[4,5-d][1,3]diazepin-8(3H)-one (9a) was followed by deprotection to 6,7-dihydro-3[(2-hydroxyethoxy)methyl]imidazo[4,5-d][1,3] diazepin-8(3H)-one (9b) and then reduction to the racemic acyclic pentostatin analogue (+/-)-3,6,7,8-tetrahydro-3-[ (2-hydroxyethoxy)methyl]imidazo[4,5-d][1,3]diazepin-8-ol (2). Ki values for the in vitro adenosine deaminase (EC 3.5.4.4; type I; calf intestinal mucosa) inhibitory activities of 1b, 1c, and 2 were determined to be 1.6 X 10(-8), 1.5 X 10(-6), and 9.8 X 10(-8) M, respectively. When compounds 2 and 9b were tested in combination with vidarabine against herpes simplex virus, type 1, in an HEp-2 plaque reduction assay, only compound 2 was able to potentiate the antiviral activity of vidarabine.

5′-Deoxy-5′-thioanalogs of adenosine and inosine 5′-monophosphate: Studies with 5′-nucleotidase and alkaline phosphatase

The interaction of 5′-deoxy-5′-thioadenosine 5′-monophosphate (A(S)MP) and 5′-deoxy-5′-thioinosine 5′-monophosphate (I(S)MP) with snake venom, 5′nucleotidase, and calf intestinal mucosa alkaline phosphatase has been characterized. The substrates, A(S)MP and I(S)MP, are analogs of adenosine 5′-monophosphate and inosine 5′-monophosphate in which sulfur replaces oxygen as the bridge between the 5′-carbon of the ribose and the phosphorous. The PS bond of both A(S)MP and I(S)MP was hydrolyzed by alkaline phosphatase producing the corresponding thionucleoside as a reaction product. The K m for A(S)MP was 270 μ m and the V for alkaline phosphatase was 110 nmol/min/mg (8% of the V for AMP), whereas the corresponding values for I(S)MP were 300 μ m and 530 nmol/min/mg protein, respectively. In contrast, 5′-nucleotidase did not catalyze hydrolysis of either A(S)MP or I(S)MP. A(S)MP and I(S)MP were competitive inhibitors of the 5′-nucleotidase hydrolysis of AMP and IMP, respectively, with K i values of 975 and 13 μ m. Decreasing the pH of the reaction from 8.1 to 7.1 lowered the K i for I(S)MP by 100-fold, to a value of 0.15 μ m.

ChemInform Abstract: BINDING SPECIFICITIES OF ADENOSINE AMINOHYDROLASE FROM CALF INTESTINAL MUCOSA WITH DIALDEHYDES DERIVED FROM HEXOFURANOSYLADENINE NUCLEOSIDES

AbstractDie Dialdehyde (I)‐(VII) werden durch Oxidation von Hexofuranosyladeninnucleosiden mit Paraperiodsäure erhalten und untersucht.

Binding specificities of adenosine aminohydrolase from calf intestinal mucosa with dialdehydes derived from hexofuranosyladenine nucleosides.

A series of nucleoside dialdehydes has been prepared as powders after treatment of hexofuranosyladenine nucleosides with paraperiodic acid; thus, periodate oxidation and purification of the products yielded dialdehydes derived from 9-(6-deoxy-beta-D-gulofuranosyl)adenine (1), 9-(6-deoxy-beta-L-gulofuranosyl)adenine (2), 9-(alpha-D-rhamnofuranosyl)adenine (3), 9-(alpha-L-rhamnofuranosyl)adenine (4), 9-(6-deoxy-alpha-L-talofuranosyl)adenine (5), 9-(5,6-dideoxy-beta-L-ribo-hex-5-enofuranosyl)adenine (6), and 9-(5,6-dideoxy-beta-D-ribo-hex-5-enofuranosyl)adenine (7). Nucleoside dialdehydes 1, 4, and 5 were weak substrates for adenosine aminohydrolase from calf intestinal mucosa. Dialdehydes 6 and 7 were not substrates for the enzyme but were rather strong competitive inhibitors, with Ki values of 50 and 7 microM, respectively. Dialdehydes 2 and 3 did not bind to the enzyme at all. The dialdehydes did not exhibit time-dependent inhibition, suggesting that they did not form covalent bonds with the protein.

Hexofuranosyladenine nucleosides as substrates and inhibitors of calf intestional adenosine deaminase

A series of hexofuranosyladenine nucleosides have been tested as substrates and inhibitors of adenosine deaminase from calf intestinal mucosa. The nucleosides differed from each other in configuration at the various carbon atoms of the hexose and had either a methyl group or hydroxymethyl group at the terminal position. It has been confirmed that the best substrates have the beta-D or alpha-L configuration at the anomeric position and an hydroxyl group on the same side of the furanose ring as adenine. However, these properties are not minimal and other nucleosides will act as substrates even if they do not have the preferred configurations or groups available. The effect of having two hydroxyl groups in the same region of the molecule and in the preferred configurations was to greatly reduce Vmax. Most structural changes resulted in changes in Vmax, whereas KM values remained fairly close. Only a change in configuration of the hydroxyl group at C-5' caused a dramatic change in affinity, as reflected in the KM. All nucleosides exhibited competitive inhibitory kinetics. In the latter studies also, a change of configuration at C-5' greatly affected binding.

Dialdehydes derived from adenine nucleosides as substrates and inhibitors of adenosine aminohydrolase.

A series of nucleoside dialdehydes have been obtained as powders after treatment of various adenine nucleosides with paraperiodic acid. Thus, oxidation gave dialdehydes derived from adenosine (1), 9-alpha-D-mannopyranosyladenine (2), 9-(5-deoxy-alpha-D-arabinofuranosyl)adenine (3), 9-alpha-L-rhamnopyranosyladenine (4), 9-beta-L-fucopyranosyladenine (5), 9-beta-D-fucopyranosyladenine (6), 9-alpha-D-arabinopyranosyladenine (7), 9-beta-D-ribopyranosyladenine (8), and 9-(5-deoxy-beta-D-erythro-pent-4-enofuranosyl)adenine (9). Nucleoside dialdehydes 1-3 and 6-8 were weak substrates for adenosine aminohydrolase from calf intestinal mucosa. Dialdehyde 8 had the strongest affinity, but 1 had the highest Vmax. All of the dialdehydes except 5 were inhibitors of the enzyme. The best inhibitors were 9 (Ki = 4 microM) and 4 (ki = 28 microM), and neither were substrates. The inhibitors did not exhibit time-dependent inhibition and did not appear to form covalent bonds with the protein. The data strongly suggest that the active form of the dialdehydes is as the open-chain dihydrates. The alcohol obtained by reduction of 9 (compound 10) was the strongest inhibitor (Ki = 0.9 microM among the related alcohols and the nucleoside dialdehydes.

Unusual substrates for adenosine deaminase from calf intestinal mucosa

A number of adenine nucleosides with exocyclic double bonds either at the 4′,5′ position in pentofuranosyl nucleosides or at the 5′,6′ position of hexofuranosyl nucleosides have been found to act as substrates for adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from calf intestinal mucosa. Most of the results obtained are contrary to the accepted minimal structural requirements for substrate activity. These nucleosides had either incorrect anomeric configurations or no hydroxyl group at C-5′ or C-3′ in the proper configuration; some compounds in corporated both structural changes. There is a possibility that the unsaturated group has a special role in binding to the enzyme.

Tight-binding inhibitors—III: A new approach for the determination of competition between tight-binding inhibitors and substrates—inhibition of adenosine deaminase by coformycin

Equations have been developed which provide the basis for the elucidation of the inhibition mechanism of a tight-binding inhibitor by studying the manner in which the substrate interferes with the bincling of the inhibitor to the enzyme. The procedure involves the determination of the pseudo-firstorder rate constants at various concentrations of the substrate and the inhibitor. This method is applicable to very tightly binding inhibitors, including irreversible inhibitors. Methods for the graphical as well as statistical analyses of the data are presented. By the application of these methods, it is demonstrated that coformycin competes with adenosine for adenosine deaminase from calf intestinal mucosa. The kinetic parameters ( ± S. E.) for the bincling of coformycin with adenosine deaminase were determined at 22° and pH 7.4; the second-order rate constant, 1.01 ( ± 0.06) × 10 60MP −1 sec −1; the first-order rate constant, 2.2 ( ± 1.0) × 10 −4 sec −1; and the dissociation constant of the EI complex, 2.2 ( ± 0.76) × 10 −10 M.

Cosolvent-buffer mixtures as models for the cytoplasmic mileu: the enzymology of adenosine aminohydrolase.

Adenosine aminohydrolase from calf intestinal mucosa is sensitive to changes in its environment produced by small mole fractions of dimethylsulfoxide (DMSO). At a mole fraction of 0.1 where the dielectric constant is lowered from that of 78 of neat water to about 76.5, Vmax was reduced by 65% and affinity for substrate (adenosine) and the two competitive inhibitors, insine and N6-benzyladenosine, was decreased markedly. However, this decreased affinity was such that Ki/Km remained virtually constant for both inhibitors. DMSO itself showed the kinetics of a mixed inhibitor with Ki decreasing with increasing mole fraction. This cosolvent also decreased the heat stability of the enzyme which suggests that enzyme conformation is altered by DMSO. Comparison of data in the presence of DMSO with previously obtained data with dioxane shows that heat stability as well as Vmax, at a given value of dielectric constant, is independent of the amount or nature of cosolvent used to achieve that dielectric constant. However, cosolvent induced changes in Ki indicate that colligative as well as dielectric constant effects contribute to the observed changes in kinetic behavior. These experiments may be considered as models for the behavior of enzymes in the medium of lowered dielectric constant expected in the vicinity of cytoplasmic membranes. The results indicate that in such an environment, adenosine aminohydrolase would be expected to be less efficient a catalyst, but equally susceptible to product inhibition, as compared to media of dielectric constant approaching that of water.