Homogenates Of Rat Intestinal Mucosa Research Articles

Intestinal absorption of aspartame decomposition products in adult rats

The dipeptide sweetener aspartame ( N- l-α-aspartyl- l-phenylalanine, 1-methyl ester; α-APM) is relatively stable in dry powder form. However, when exposed to elevated temperature, extremes of pH, and/or moisture, α-APM is converted into a variety of products. In aqueous solution α-APM decomposes to yield methanol, two isomeric forms of l-aspartyl- l-phenylalanine (Asp-Phe) [α-Asp-Phe and β-Asp-Phe], and APM's diketopiperazine cyclo-Asp-Phe. Depending on beverage storage conditions, individuals drinking α-APM-sweetened beverages may consume small quantities of these three compounds. Relatively little has been published about the metabolism of β-Asp-Phe and cyclo-Asp-Phe. We compared the absorption and metabolism of α-Asp-Phe, β-Asp-Phe, and cyclo-Asp-Phe with that of l-phenylalanine (Phe) in adult rats. Steady-state perfusion studies of rat jejunum indicated rapid carrier-assisted uptake of Phe and α-Asp-Phe, but only slow passive diffusion of β-Asp-Phe and cyclo-Asp-Phe from the lumen. Homogenates of rat intestinal mucosa, liver, and cecal contents, as well as homogenates of pure cultures of Escherichia coli B, catalyzed the hydrolysis of α-Asp-Phe, but not cyclo-Asp-Phe. Homogenates of E coli and rat cecal contents, but not homogenates of rat liver or intestinal mucosa catalyzed the hydrolysis of β-Asp-Phe.

Stimulation of mucosal bicarbonate secretion in rat duodenum in vivo by BW755C.

Bicarbonate secretion from 12 mm segments of duodenum just distal to the Brunner's gland area was titrated (pH 7.60) in situ in anesthetized rats. Intravenous BW755C (10-20 mg/kg) increased both bicarbonate secretion and the transmucosal electrical potential difference and pretreatment with indomethacin (3 mg/kg intravenously) prevented these effects. Indomethacin also inhibited stimulation of HCO3- secretion by luminal acid (10 mM HCl) but had no effect on the rise in secretion in response to exogenous (luminal) prostaglandin E2. The results support previous suggestions of a role for endogenous prostaglandins in mediation of the HCO3- response to acid and are consistent with the recent demonstration that BW755C increased prostaglandin formation in homogenates of rat intestinal mucosa. Stimulation of HCO3- secretion by BW755C was not enhanced but attenuated by preexposure to luminal acid, suggesting that the latter increases secretion by effects other than mucosal mobilization of arachidonate.

An examination of the stereochemical course of acylation of 2-monoacylglycerols by rat intestinal villus cells using [ 2H 3]palmitic acid

The stereochemical course of acylation of 2-monoacylglycerols by rat intestinal mucosa was investigated using isolated villus cells with 2-lauroylglycerol and [ 2H 3]palmitic acid as substrates. The newly synthesized X-1,2-diacylglycerols and triacylglycerols were recognized on the basis of the content, positional distribution and molecular association of the fatty acids by gas-liquid chromatography/mass spectrometry in combination with stereospecific analysis. It was found that the free X-1,2-[ 2H 3] palmitoyllauroylglycerols contained 74% sn-1,2- and 26% sn-2,3-enantiomers, which were utilized for triacylglycerol formation in the same proportion. Some of the 2-monolauroylglycerol became hydrolyzed and the released lauric acid was utilized along with [ 2H 3]palmitic acid for diacylglycerol and triacylglycerol formation via both the phosphatidic acid and the 2-monoacylglycerol pathway. The contribution of the phosphatidic acid pathway (12–16%) was determined by estimating the relative proportion of the newly synthesized triacylglycerols containing [ 2H 3]palmitic acid in the sn-2 position. After correcting for the contribution of the phosphatidic acid pathway, the sn-1,2-/ sn-2,3-enantiomer ratio in the X-1,2-[ 2H 3] palmitoyllauroylglycerol was estimated to be 71/29. These results are consistent with those previously obtained with glycerol-labelled 2-mono-acylglycerols and intact tissue or homogenates of rat intestinal mucosa. It is suggested that the 2-mono-acylglycerol transacylase, if a single enzyme, possesses a low stereospecificity, or that two enzymes exist of unequal concentration and /or activity.

Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa. Subcellular localization and temperature stability.

The demonstration of the ornithine biosynthesis from glutamate in cell-free homogenates of rat intestinal mucosa by Ross, G., Dunn, D., and Jones, M.E. (1978) Biochem. Biophys. Res. Commun. 85, 140-147 suggested that this tissue might have the capacity to convert glutamate to pyrroline-5-carboxylate (P5C). We have shown in the preceding paper (Wakabayashi, Y., and Jones, M.E. (1983) J. Biol. Chem. 258, 3865-3872) that this is the case. The intracellular distribution of the P5C-synthesizing activity was investigated utilizing a newly developed procedure for subcellular fractionation of the rat intestinal mucosa. We found that the activity resided in the mitochondrial fraction as characterized by marker enzymes and an electron micrograph. The mitochondrial membrane fraction, freed of the soluble matrix and intermembrane space enzymes, retained all of the P5C-synthesizing activity. Addition of the soluble fraction to the membrane fraction did not affect the activity. P5C synthase, the name we have chosen for the protein(s) that catalyzes P5C synthesis from glutamate when ATP and NADPH are present, is susceptible to thermal inactivation in the presence of detergent. By lowering the incubation temperature to or below 20 degrees C, one can obtain a linear production of P5C with respect to time and protein concentration. Lower incubation temperatures are recommended for routine assay of this enzyme(s). Addition of 30% glycerol to the incubation mixture resulted in a linear formation of P5C with time at 30 degrees C; this and other data suggest that polyhydroxylic compounds may protect this protein against denaturation. Preliminary experiments suggest that P5C synthase can be extracted from a mitochondrial membrane in the presence of detergent, a high salt concentration, and glycerol. The possibility that the enzyme(s) is located in the inner mitochondrial membrane is discussed.

Immobilized Soybean Trypsin Inhibitor in the Stabilization, Resolution and Purification of Adenosine‐3′: 5′‐monophosphate‐Dependent Protein Kinases

The protein kinase dependent on adenosine 3′: 5′‐monophosphate (cAMP) in homogenates of rat intestinal mucosa was found to be remarkably unstable during its purification due (at least in part) to proteolysis. Since soybean trypsin inhibitor was found to protect the kinase from inactivation, an attempt was made to use this inhibitor, coupled to Sepharose, in order to extract and remove the proteolytic enzyme(s) responsible for the instability at an early stage of the purification. Indeed, this column excluded the undissociated form of cAMP‐dependent protein kinase (R2C2) in a considerably more stable form, illustrating the use of immobilized proteolytic inhibitors for stabilizing proteins in crude extracts containing potent proteases. But unexpectedly the same column retarded a protein kinase which was not activated by cAMP. The latter enzyme was identified as the free catalytic subunit (C) of cAMP‐dependent protein kinase since it was found to be inhibited by the Walsh‐Krebs specific protein inhibitor. On the basis of these observations, a method was developed for the purification of C from rabbit muscle which is based on (a) passage of the crude extract on the column and (b) biospecific dissociation of R2C2 by cAMP and rechromatography of the extract on the same column. This procedure yields > 300‐fold purification of C in the last step (with a recovery of 34%) and a rapid resolution between R2C2 and C. The same column can also be used for the resolution of type I and type II cAMP‐dependent protein kinases, and for > 40 fold purification of the type II form of the enzyme, in one step. The theoretical and practical implications of these results with regard to the use and abuse of biorecognition in chromatography are discussed.

Kinetics of phosphoglucose ester hydrolysis by rat intestinal mucosa.

SummaryThe enzymatic hydrolysis of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) by homogenates of rat intestinal mucosa was quantitated. These substrates were not utilized by enzymes other than phosphatases to any appreciable extent. The mean Km value for the hydrolysis of G6P was higher (4.3 mM) than that for G1P (1.69 mM). A variability was observed in Km values in each substrate group. However, the Km/Vmax ratio was constant for each of the two substrates. Such proportional changes in Km and Vmax values allow the approximation of the rate constant (K1) related to the formation of the enzyme-substrate complex. The K1 value thus estimated for G1P was approximately 1.68 times that for G6P.This work was supported by U. S. Army Research and Development Command, Department of the Army, under Research Contract No. DA-49-193-MD-2415, and by Research Grant AM 11945 from the National Institutes of Health, U. S. Public Health Service.

Intestinal absorption of stereoisomers of dipeptides in the rat.

1. Studies have been made of jejunal absorption rates in vivo in the rat of the stereoisomers of alanylphenylalanine, leucyl-leucine and glycyltryptophan. Absorption rates of l-alanyl-l-phenylalanine were about 200 times those of d-alanyl-d-phenylalanine, l-leucyl-l-leucine about 24 times those of the dd-isomer, and glycyl-l-tryptophan 5 times those of glycyl-d-tryptophan. The mixed ld- and ld-isomers were absorbed at intermediate rates. 2. Absorption rates were positively correlated with the rate of hydrolysis of each dipeptide by homogenates of rat intestinal mucosa. The transport rate and rate of hydrolysis of glycyl-d-tryptophan, d-alanyl-d-phenylalanine and d-leucyl-d-leucine were significantly greater in the ileum than in the jejunum. 3. When given by stomach tube the most slowly absorbed dipeptides, d-alanyl-d- phenylalanine, d-leucyl-d-leucine and glycyl-d-tryptophan were the only ones to be excreted in significant amounts in the urine, showing that they were absorbed as the entire molecule and were resistant to hydrolysis by tissue peptidases. 4. There was a close inverse correlation between the rates of transport by rat jejunum of glycine, d-alanine, d-leucine, d-phenylalanine, d-tryptophan, d-alanyl-d- phenylalanine, d-leucyl-d-leucine and glycyl-d-tryptophan and the molecular weight of each compound, suggesting that diffusion mechanisms play an appreciable part in jejunal absorption of these compounds. No such correlation was found in the case of the ileum.

Studies on zinc absorption: 65Zn binding by homogenates of rat intestinal mucosa.

Approximately 60% of the 65Zn in labeled homogenates of rat intestinal mucosa was recovered in a 45,000g supernatant, and about 50% of the counts in this supernatant could be recovered in a 45-80% ammonium sulfate precipitate. The bound 65Zn in the ammonium sulfate precipitate could be resolved into two peaks by gel filtration on Sephadex G-100. The first radioactive fraction consisted of substances having molecular weights of 150,000 or more, and the second tion consisted of substances having molecular weights in the range of 10 to 15,000. Electrophoresis of the material from the first Sephadex peak always yielded 10 or more visible protein bands; whereas material from the second peak yielded either 3 or 4 visible bands. It was not possible to determine which, if any, of the protein bands were originally labeled, as the 65Zn dissociated during electrophoresis and migrated to the cathode. To date, there is no evidence to link any of these compounds with intestinal absorption of zinc.

The Enzymatic Reduction of Retinal to Retinol in Rat Intestine

Abstract The reduction of retinal to retinol was examined with enzyme preparations from homogenates of rat intestinal mucosa. Retinal reduction was catalyzed by a soluble mucosal enzyme which was purified 13-fold by ammonium sulfate precipitation, column chromatography, and heating (55° for 6 min). The enzyme was relatively heat-stable and had a molecular weight approximately in the range of 60,000 to 80,000. The partly purified reductase was unable to oxidize ethanol in the presence of NAD+. Retinal reduction required NADH or NADPH as cofactor. Both reduced nucleotides were effective; at low nucleotide concentration NADH was more effective, whereas at high concentrations the reaction rate was slightly greater with NADPH. The reaction was stimulated by the addition of glutathione and was inhibited by —SH inhibitors. No other cofactors were required. There was a sharp pH optimum near 6.3. Retinal reduction displayed typical Michaelis kinetics, with a Vmax of 2 x 10-6 moles of retinal formed per hour per mg of protein and with an apparent Km of 2 x 10-5 m. The enzyme appears to be a relatively nonspecific aldehyde reductase. Short and medium chain aliphatic aldehydes, of length C-2 to C-14, were actively reduced, with greatest activity being seen with aldehydes of length C-4 to C-8. Unsaturated C-18 fatty aldehydes were reduced at a lesser rate, but saturated aldehydes of length C-16 or greater were not reduced. The enzyme was stereospecific for 4R-NADH-4-3H1, and did not incorporate tritium from 4S-NADH-4-3H1 into the product retinol.

Sulfate metabolism in the intestinal mucosa of weanling and adult rats.

SummarySulfate metabolism in homogenates of rat intestinal mucosa was investigated in the jejunal and ileal segments. Direct comparisons were made between activities of homogenates of adult (43-day-old) and suckling (13-day-old) rats. The hydrolysis of p-nitrocatechol sulfate or p-nitrophenyl sulfate proceeded most rapidly in incubation mixtures which contained homogenates of ileal mucosa from 13-day-old rats. Sulfokinase activity was observed to be highest in homogenates of jejunal segments from suckling animals. In both cases the differences were marked, and may suggest important developmental changes in physiological function of these two segments of the gastrointestinal tract.

Mechanism of the Biosynthesis of Vitamin A from β-Carotene

Experiments were conducted to determine whether the hydrogen atoms attached to the 2 central carbon atoms of β-carotene are retained or are lost during the conversion of β-carotene to vitamin A. Doubly labeled β-carotene, uniformly labeled with 14C throughout the molecule but specifically labeled with 3H only at the central carbon atoms (C-15 and C-15'), was fed to each of two lymph fistula rats. Lymph was collected and the retinyl esters isolated. The ratio of 3H to 14C in the retinyl esters from lymph, and in the retinol obtained from these esters by saponification, was identical with the ratio of 3H to 14C in the dietary β-carotene. This indicated that there was no loss of 3H relative to 14C during the conversion of β-carotene to vitamin A. Complete retention of 3H relative to 14C was also found during the conversion in vitro of doubly labeled β-carotene to retinal with homogenates of rat intestinal mucosa. The hydrogen atoms attached to the central carbon atoms of β-carotene must therefore be retained completely during the biosynthesis of vitamin A. In order to verify that the 3H label was attached only to the central carbon atoms of β-carotene, the retinol (from lymph) was oxidized to retinoic acid by a two-step procedure. This oxidation was accompanied by a complete loss of 3H label from the molecule. The biosynthesis of vitamin A from β-carotene is most likely a dioxygenase reaction, in which molecular oxygen reacts with the 2 central carbon atoms of β-carotene, followed by the cleavage of the central double bond of β-carotene to yield 2 molecules of retinal.

BIOSYNTHESIS OF VITAMIN A WITH RAT INTESTINAL ENZYMES.

Vitamin A is synthesized from beta-carotene in cell-free homogenates of rat intestinal mucosa, the biosynthetic enzymatic activity being present in the soluble protein fraction of the homogenate. Also required are a heat-stable factor in the particulate fraction, molecular oxygen, and bile salts. The reaction is stimulated by glutathione. The product, obtained in yields of up to 50 percent, has been identified as vitamin A aldehyde (retinal) by way of its semicarbazone derivative. The reaction mechanism involves the central cleavage of beta-carotene into two molecules of retinal.

THE REDUCTION OF INORGANIC SULPHATE TO INORGANIC SULPHITE IN THE SMALL INTESTINE OF THE RAT.

1. Whole scrapings of rat intestinal mucosa were incubated with carrier-free sodium [(35)S]sulphate. Radioactivity was found in S-sulphocysteine and to a small extent in S-sulphoglutathione. 2. Whole scrapings of rat intestinal mucosa incubated with carrier-free sodium [(35)S]sulphate and oxidized glutathione formed S[(35)S]-sulphoglutathione as the main radioactive product. The amount of S[(35)S]-sulphocysteine formed was considerably lower than in a control that contained no oxidized glutathione. 3. The supernatant fraction of homogenates of rat intestinal mucosa catalyses the NADPH-dependent reduction of adenosine 3'-phosphate 5'-sulphatophosphate to inorganic sulphite. NADH or GSH fail to replace NADPH as reducing agents. 4. The formation of inorganic [(35)S]sulphite from inorganic [(35)S]-sulphate may account for the incorporation of [(35)S]sulphate into S-sulphoglutathione by the small intestine of the rat in vivo and in vitro.

Specificity of an Intestinal Lipase for Monoglycerides.

Abstract : An enzyme found in the homogenates of rat intestinal mucosa has been demonstrated to be specific for the hydrolysis of 1- and 2-monoglycerides. During a two hour incubation at 37 C, both 1- and 2-monoglycerides are hydrolyzed to a comparable extent (17-19%). Under the same conditions, diand triglycerides undergo little or no hydrolysis. This enzyme appears to be different from most enzymes previously reported in the intestinal mucosa or succus entericus. The enzymes previously reported have their major activity directed toward the hydrolysis of triglycerides only or hydrolyze mono-, di-, and triglycerides at approximately equal rates. The exact role of this monoglyceride specific lipase is difficult to delineate. Since pancreatic lipase is specific for the hydrolysis of the 1- and 3-positions of triglycerides, this enzyme may have a definite role in the completion of normal digestion of triglycerides. (Author)

The hexokinase activity of the intestinal mucosa

Homogenates of rat intestinal mucosa phosphorylate glucose, fructose, mannose, glucosamine, 2-deoxyglucose, allose and l-sorbose. No phosphorylation of galactose or 3-methylglucose has been observed. With the exception of l-sorbose, the phosphorylations are carried out by a single enzyme with a pattern of specifictty similar to that of brain hexokinase. The hexoses appear to be phosphorylated in the 6 position. Glucose-6-phosphate inhibits the enzyme. The data indicate that hexokinase is not a part of the mechanism of intestinal absorption of sugars.