Some properties of phosphodiesterase I from microsomes of rat intestinal mucosa

1970 ◽  
Vol 48 (10) ◽  
pp. 1151-1159 ◽  
Author(s):  
I. Hynie ◽  
S. H. Zbarsky
Keyword(s):  
Intestinal Mucosa ◽  
Assay System ◽  
Noncompetitive Inhibition ◽  
Optimum Ph ◽  
Nitrophenyl Ester

Some properties of phosphodiesterase I from microsomes of rat intestinal mucosa were studied using the p-nitrophenyl ester of thymidine 5′-phosphate as substrate. The optimum pH was found to be 9.5–9.6 in several buffer systems. The activity was destroyed at temperatures of 63–68°, depending on the pH of the medium. Mg2+, Ca2+, Sr2+, Ba2+, and Ni2+ had a variable activating effect while Al3+, Cu2+, and Be were inhibitory. The enzyme was inhibited completely by 10−5 M EDTA or EGTA in the assay system used. The evidence obtained suggested phosphodiesterase I is a metal-requiring enzyme. Glycine, bicine, and tricine inhibited the enzyme at higher concentrations. Cysteine, 2-mercaptoethanol, and dithiothreitol were strongly inhibitory at a concentration of 10−3 M. The Km of the enzyme was 8.5 × 10−4 M. In 6 M urea 40% of the activity of the enzyme was lost irreversibly after 24 h at 4°. In 3 M urea there was an immediate and reversible mixed competitive–noncompetitive inhibition and the Km was increased seven to eight times.

scholarly journals The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

1972 ◽  
Vol 128 (3) ◽  
pp. 579-586 ◽  
Author(s):  
A. Sheltawy ◽  
M. Brammer ◽  
D. Borrill
Keyword(s):  
Guinea Pig ◽  
Subcellular Fractions ◽  
Assay System ◽  
Acid Phosphatases ◽  
Alkaline Phosphatases ◽  
Pig Brain ◽  
Optimum Ph ◽  
Ph Range ◽  
Guinea Pig Brain ◽  
Optimum Ph Range

1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7–7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has Km1.43×10-4m. 2. A factor from the ‘pH5 supernatant’ of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ‘pH5 supernatant’. Maximum stimulation was observed with the P1 fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ‘pH5 supernatant’. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5′-nucleotidase, but different from that of alkaline phosphatase.

scholarly journals A new assay procedure for monoglyceride acyltransferase

1974 ◽  
Vol 141 (2) ◽  
pp. 407-411 ◽  
Author(s):  
Verena J. Short ◽  
David N. Brindley ◽  
Raymond Dils
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Microsomal Fraction ◽  
Assay System ◽  
Acyltransferase Activity ◽  
Palmitoyl Carnitine ◽  
Assay Procedure ◽  
Acyl Acceptors

1. A new assay system is described for monoglyceride acyltransferase (acylglycerol palmitoyltransferase, EC 2.3.1.22) in which palmitoyl-CoA is generated from palmitoyl-(-)-carnitine. 2. With the microsomal fraction from homogenates of guinea-pig intestinal mucosa, the Vmax. of this enzyme decreased with different acyl acceptors in the order 2-monopalmitoylglycerol>2-hexadecylglycerol>rac-1-monopalmitoylglycerol. 3. There were highly significant correlations between the monoglyceride acyltransferase activity as measured with these three substrates. This demonstrates that each of these substrates can be used to measure the same enzyme activity. 4. The advantages of using generated palmitoyl-CoA with 2-hexadecylglycerol and rac-1-monopalmitoylglycerol as model substrates for this enzyme are discussed.

scholarly journals Vitamin A and carotenoids. The enzymic conversion of β-carotene into retinal in hog intestinal mucosa

1969 ◽  
Vol 114 (4) ◽  
pp. 689-694 ◽  
Author(s):  
Noel H. Fidge ◽  
Frank Rees Smith ◽  
DeWitt S. Goodman
Keyword(s):  
Intestinal Mucosa ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Enzyme Preparations ◽  
Optimum Ph ◽  
Tween 40 ◽  
Ph Range ◽  
Optimum Ph Range ◽  
Β Carotene

The conversion of β-carotene into retinal was studied in vitro with enzyme preparations from homogenates of hog intestinal mucosa. The hog mucosal enzyme was purified about 27-fold by precipitation with ammonium sulphate, chromatography on DEAE-Sephadex and gel filtration on Sephadex G-200. The reaction displayed a narrow optimum pH range (approx. 7·8–8·2). The enzyme was stimulated strongly by the addition of thiols, and was inhibited by thiol inhibitors and by the chelating agents αα′-bipyridyl and o-phenanthroline. The reaction required the addition of an appropriate detergent (or bile salt); maximal activity was obtained by addition of an appropriate combination of detergents and lipid (specifically Tween 40, sodium glycocholate and sphingomyelin). The reaction displayed Michaelis kinetics with Km1·3×10−6m and Vmax.1·1nmole of retinal formed/hr. (for 0·7mg. of enzyme protein). The properties of the hog enzyme are similar to those previously reported for a less purified rat enzyme preparation.

Cl- -HCO3- -stimulated ATPase in intestinal mucosa of Aplysia

1985 ◽  
Vol 248 (2) ◽  
pp. R241-R248 ◽  
Author(s):  
G. A. Gerencser ◽  
S. H. Lee
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Intestinal Mucosa ◽  
Membrane Fraction ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Disulfonic Acid ◽  
Differential Centrifugation ◽  
Optimum Ph

The serosa negative transepithelial potential difference across Aplysia intestine is generated by a Na+-independent, active electrogenic Cl- absorptive mechanism. In an attempt to clarify the Cl- absorptive mechanism an anion-stimulated ATPase was prepared from plasma membranes from Aplysia enterocytes utilizing differential centrifugation and sucrose density gradient techniques. ATPase activity, which could be activated by either Cl- or HCO3-, was found in the plasma membrane fraction. Maximal anion-ATPase activity was achieved with either 25 mM Cl- or 25 mM HCO3-. The apparent Km for Cl- activation of the ATPase was 10.3 mM, whereas apparent Km for HCO3- was 9.7 mM. ATP was the most effective nucleotide substrate for both HCO3- and Cl- -ATPase activities, whereas optimum pH for both activities was 7.8. These enzyme activities were inhibited more than 30% by thioacyanate (10 mM). Acetazolamide and vanadate were also found to strongly inhibit both Cl- and HCO3- -ATPase activities, whereas 10 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 1 mM furosemide, or 1 mM ouabain had little or no effect. These results are consistent with the hypothesis that the active Cl- transport mechanism in Aplysia intestine could be a Cl- -HCO3- -stimulated ATPase found in the enterocyte plasma membrane.

A Study of the Proteolytic Enzyme Activity of the Pyloric Caeca of Redfish

1953 ◽  
Vol 10 (8) ◽  
pp. 590-598 ◽  
Author(s):  
Joseph A. Stern ◽  
Ernest E. Lockhart
Keyword(s):  
Enzyme Activity ◽  
Proteolytic Activity ◽  
Intestinal Mucosa ◽  
Proteolytic Enzyme ◽  
Enzyme Preparation ◽  
Statistical Study ◽  
Optimum Temperature ◽  
Suitable Material ◽  
Pyloric Caeca ◽  
Optimum Ph

The proteolytic activity of an enzyme preparation from the pyloric caeca of redfish (Sebastes marinus) was studied and measured colorimetrically by the biuret reaction. The optimum temperature of this preparation was found to be 51–52 °C. A statistical study of the data showed the optimum pH to be 8.75, or slightly higher than the optimum pH of trypsin. A comparison of the actions of a commercial leather bate, hog intestinal mucosa, papain, pancreatin, trypsin and the caecal enzyme of redfish on casein led to the conclusion that the pyloric caeca of redfish would furnish a suitable material from which to prepare a leather bate.

Activation of isolated heterophils from chicks stimulated in vivo with salmonella enteritidis-immune lymphokines

1996 ◽  
Vol 54 ◽  
pp. 760-761
Author(s):  
R. B. Moyes ◽  
R. E. Droleskey ◽  
M. H. Kogut ◽  
J. R. DeLoach
Keyword(s):  
Lamina Propria ◽  
Intestinal Mucosa ◽  
Salmonella Enteritidis ◽  
Intestinal Tract ◽  
Polymorphonuclear Cells ◽  
Subclinical Infection ◽  
Egg Products ◽  
Immune Lymphokines ◽  
Organ Invasion

Salmonella enteritidis (SE) is of great concern to the poultry industry due to the organism's ability to penetrate the intestinal mucosa of the laying hen and subsequently colonize the ovaries and yolk membrane. The resultant subclinical infection can lead to SE infection of raw eggs and egg products. Interference with the ability of the organism to invade has been linked to the activation and recruitment of inflammatory polymorphonuclear cells, heterophils, to the lamina propria of the intestinal tract.Recently it has been established that heterophil activation and increased resistance to SE organ invasion can be accomplished by the administration of SE-immune lymphokines (SE-ILK) obtained from supernatants of concanavalin-A stimulated SE immune T lymphocytes from SE hyperimmunized hens. Invasion of SE into the lamina propria provides a secondary signal for directing activated heterophils to the site of SE invasion.

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