Subcellular Distribution of Hydrolase Activities for Glycine and Leucine Homopeptides in Human Jejunum

1978 ◽  
Vol 54 (2) ◽  
pp. 205-207
Author(s):  
J. A. Nicholson ◽  
T. J. Peters
Keyword(s):  
Brush Border ◽  
Subcellular Distribution ◽  
Brush Border Membrane ◽  
Specific Marker ◽  
Peptidase Activity ◽  
Small Peptides ◽  
Normal Human ◽  
Analytical Fractionation ◽  
Hydrolysis Of ◽  
Isopycnic Centrifugation

1. The subcellular distribution of peptidase activities in the normal human jejunum against glycine and leucine homopeptides has been investigated with an analytical fractionation technique. 2. An 8000 g-min supernatant was prepared from homogenates of Crosby capsule biopsy specimens and subjected to isopycnic centrifugation in a Beaufay automatic zonal rotor. 3. The distribution of subcellular organelles in the gradient was established by measurement of organelle-specific marker enzymes. 4. A sensitive fluorimetric assay for glycine peptidase was developed and used for the localization of peptidase activity with peptides composed of from two to five glycine residues as substrates. 5. Glycine peptidase activity was located in the cytosol and in the brush-border membrane but the distribution of activity varied markedly with the chain-length of substrate; the longer the peptide the greater the proportion of activity associated with the brush border. Leucine peptidase showed a similar variation in cytosol—brush border distributions. 6. The results are consistent with concepts that suggest absorption and intracellular hydrolysis of small peptides and brush-border digestion of larger peptides.

Effect of ethanol on peptidases of hamster jejunal brush-border membrane

1982 ◽  
Vol 242 (5) ◽  
pp. G442-G447
Author(s):  
P. K. Dinda ◽  
I. T. Beck
Keyword(s):  
Conformational Changes ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
Time Dependent ◽  
Peptidase Activity ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Hydrolysis Of

This study was undertaken to investigate the effect of ethanol on the brush-border activity of the small intestine. Brush-border membrane isolated from hamster jejunum was incubated with L-phenylalanylglycine (Phe-Gly), L-leucylglycine (Leu-Gly), or glycyl-L-tyrosine (Gly-Tyr) in the absence and presence of 1-5% (wt/vol) ethanol, and the L-amino acids liberated were determined. Ethanol was found to depress the hydrolysis of all peptides in a dose-dependent manner. The inhibitory effect of ethanol on the peptidases does not appear to be time dependent. The ethanol-induced inhibition of peptidase activity is completely reversible. Kinetic studies indicate that ethanol caused a decrease in the Vmax of the enzymes responsible for the hydrolysis of the Phe-Gly and Gly-Tyr but did not have any effect on their Km. In the hydrolysis of Leu-Gly, two enzymes were involved, and ethanol depressed the Vmax of both without affecting the Km of either. These findings suggest that ethanol produces conformational changes of the peptidases involved in the hydrolysis of these three dipeptides.

scholarly journals Different handling of parathyrin by basal-lateral and brush-border membranes of the bovine kidney cortex

1980 ◽  
Vol 188 (3) ◽  
pp. 649-656 ◽  
Author(s):  
H Mohr ◽  
R D Hesch
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Kidney Cortex ◽  
Small Peptides ◽  
Uniform Size ◽  
Bovine Kidney ◽  
Brush Border Membranes ◽  
Membrane Bound ◽  
Uniform Size Distribution

The two parts of the bovine kidney cortex plasma membrane, the basal-lateral and the brush-border membrane, were simultaneously prepared from the same organ. Both types of membrane bound parathyrin, but only from the basal-lateral fraction was the hormone displaceable by its bioactive N-terminal fragment. In parallel, parathyrin-stimulated adenylate cyclase was predominantly found in basal-lateral membranes. The hormone was fragmented by both membrane types. Basal-lateral membranes generated fragments with a rather uniform size distribution (somewhat smaller than the intact peptide) and apparently preferred the hormone itself as a substrate. In contrast, the fragments produced by brush-border membranes were numberous small peptides.

scholarly journals A comparison of the active site of maltase-glucoamylase from the brush border of rabbit small intestine and kidney by chemical modification studies

1991 ◽  
Vol 274 (2) ◽  
pp. 349-354 ◽  
Author(s):  
B Pereira ◽  
S Sivakami
Keyword(s):  
Chemical Modification ◽  
Brush Border ◽  
Active Site ◽  
Brush Border Membrane ◽  
Kidney Enzyme ◽  
Rabbit Intestine ◽  
Rabbit Small Intestine ◽  
Intestinal Enzyme ◽  
The One ◽  
Hydrolysis Of

The neutral maltase-glucoamylase complex has been purified to homogeneity from the brush-border membrane of rabbit intestine and kidney. Chemical modification of the amino acid side chains was carried out on the purified enzymes. Studies on the kidney enzyme revealed that tryptophan, histidine and cysteine were essential for both maltase and glucoamylase activities, whereas tryptophan, histidine and lysine were essential for the maltase and glucoamylase activities of the intestinal enzyme. Though there was no difference in the amino acids essential for the hydrolysis of maltose and starch by any one enzyme, starch hydrolysis seems to require two histidine residues instead of the one which is required for maltose hydrolysis. This appears to be true for both the intestinal and kidney enzymes.

The Effects of Prednisolone on the Rat Enterocyte at a Subcellular Level

1978 ◽  
Vol 55 (5) ◽  
pp. 435-443
Author(s):  
R. M. Batt ◽  
G. Wells ◽  
T. J. Peters
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Equilibrium Density ◽  
Proximal Jejunum ◽  
Brush Border Enzymes ◽  
Brush Borders ◽  
Brush Border Enzyme ◽  
Mitochondrial Malate Dehydrogenase ◽  
Subcellular Level ◽  
Isopycnic Centrifugation

1. Enterocytes, isolated from the proximal jejunum and distal ileum of normal and prednisolone-treated rats, were homogenized and fractionated by isopycnic centrifugation on sucrose density gradients. The distributions of marker enzymes for the principal subcellular organelles, RNA and protein were determined and related to the activities per enterocyte. 2. In enterocytes from the jejunum and ileum of prednisolone-treated animals the activities of particulate brush-border enzymes and of both soluble and mitochondrial malate dehydrogenase were increased compared with those of the control system. The equilibrium density of the brush borders was enhanced in the prednisolone-treated jejunum. The modal densities of the other organelles were unaltered by prednisolone administration. 3. There was a large increase in the total RNA content of enterocytes from the jejunum and ileum of prednisolone-treated animals. This was predominantly associated with a distinct particulate component, indicative of a proliferation of the rough endoplasmic reticulum and consistent with an enhanced rate of protein synthesis. 4. Studies of latent brush-border enzyme activities, the mechanical fragility of isolated brush borders and electron microscopy suggest that steroid administration results in no marked alterations in the gross conformation of the brush- border membrane or in the orientation of the enzymes within the membrane.

Enhancement of brush border membrane peptidase activity in rat jejunum induced by starvation

2000 ◽  
Vol 440 (1) ◽  
pp. 75 ◽  
Author(s):  
Takashi Ihara ◽  
Tomoyuki Tsujikawa ◽  
Yoshihide Fujiyama ◽  
Hisao Ueyama ◽  
Iwao Ohkubo ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Peptidase Activity ◽  
Rat Jejunum

scholarly journals Peptidase Activity of Amidinated Thrombin

1977 ◽  
Author(s):  
E.P. Kang
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Enzymic Activity ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
Human Thrombin ◽  
Small Change ◽  
Hydrolysis Of ◽  
Hydrolysis Activity

Human thrombin, free of plasminogen and plasmin, was treated with ethyl acetimidate hydrochloride in order to modify the lysyl residues of the protein. By monitoring the enzymic activity in the modification mixture, it was found that the reaction was completed in about one hour and the loss of activity of thrombin was proportional to the amount of modification. After the removal of the excess ethyl acetimidate, approximately 25% of the clotting activity and of the hydrolysis activity for small peptides remained. Amino acid analysis of this modified thrombin indicated about 80% of the lysyl residues had been modified with no apparent change of other amino acid residues. By studying the thrombolytic hydrolysis of Bz-phe-val-arg-pNA, the kcat of the amidinated thrombin was about 8% of the control while the KM Secreased to 0.056 μM from 0.098 μM. The modification of the lysyl residues of thrombin, therefore, has lowered the catalytic efficiency of the enzyme with a rather small change in binding affinity. This suggests that modification of lysyl residues in the neighborhood of the active site hinders the catalytic hydrolysis of the small peptides.

Evidence that L-sucrose is resistant to hydrolysis catalyzed by jejunal brush border enzymes

1982 ◽  
Vol 60 (5) ◽  
pp. 652-654 ◽  
Author(s):  
P. K. Dinda ◽  
I. T. Beck ◽  
Walter A. Szarek ◽  
George W. Hay ◽  
Edward R. Ison ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Liquid Chromatograph ◽  
Brush Border Enzymes ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border ◽  
Hydrolysis Of

The sucrase–isomaltase complex of the intestinal brush border membrane (BBM) catalyzes the hydrolysis of sucrose. The stereospecificity of this enzyme, however, is not known. To investigate this, BBM of hamster jejunum was incubated with D-sucrose or L-sucrose, and the reaction mixture was analyzed using a gas–liquid chromatograph. It was found that D-sucrose was hydrolyzed to its monomers, but L-sucrose remained unhydrolyzed. It is concluded that the sucrase–isomaltase of intestinal BBM of hamster jejunum does not hydrolyze L-sucrose and therefore this enzyme is stereospecific.

Proteins and enzymes of the brush-border membrane of mouse intestine: influence of organ culture on gel electrophoretic patterns

1982 ◽  
Vol 60 (4) ◽  
pp. 434-443 ◽  
Author(s):  
A. Berteloot ◽  
J. S. Hugon
Keyword(s):  
Membrane Proteins ◽  
Organ Culture ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Specific Activity ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Marker

Purifications of mouse intestinal brush-border membranes from control explants and scrapings of intestinal mucosa have been compared. Based on the specific activity of sucrase used as a specific marker of these membranes, higher purification factors were obtained with control explants (24.7 ± 0.9) as compared with scrapings of intestinal mucosa (14.8 ± 0.9). However, similar patterns of proteins and enzymes were obtained by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis after membrane solubilization by 2% SDS at room temperature. After 24 h of culture, higher molecular weight species of maltase–glucoamylase–isomaltase (band 4), alkaline phosphatase (bands 9–10), and trehalase (band 17) have been observed. Enzyme species appearing in the particulate fraction of culture media were, however, identical with those found at the brush-border membrane level in control explants, except for trehalase. These results are interpreted by considering the possible adsorption of serum components to brush-border membrane proteins. It thus appears that the membrane proteins and enzymes released in the media during organ culture are identical with those synthesized in the tissue in vitro or in vivo.

Active transport of dopamine in human placental brush-border membrane vesicles

1992 ◽  
Vol 262 (5) ◽  
pp. C1189-C1196 ◽  
Author(s):  
S. Ramamoorthy ◽  
F. H. Leibach ◽  
V. B. Mahesh ◽  
V. Ganapathy
Keyword(s):  
Transport System ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Dopamine Uptake ◽  
Negative Membrane Potential ◽  
Normal Human ◽  
Monoamine Uptake Inhibitors ◽  
Menten Constant

Brush-border membrane vesicles isolated from normal human term placentas were found to accumulate dopamine against a concentration gradient when an inwardly directed NaCl gradient was imposed across the membrane. The activity of the transport system was obligatorily dependent on Na+ as well as Cl-. Intravesicular H+ and intravesicular K+ stimulated the transport activity. The system possessed high affinity for dopamine and norepinephrine, with considerably lower affinity for serotonin. The stoichiometry of Na(+)-Cl(-)- dopamine was 1:1:1. The system was electrogenic because the NaCl-dependent dopamine uptake was stimulated by an inside-negative membrane potential, and this characteristic was observed in the presence and in the absence of intravesicular K+. Kinetic analysis revealed that the uptake was due to a carrier-mediated component plus a diffusion/binding component. The apparent Michaelis-Menten constant for dopamine for the carrier-mediated component was 19 +/- 7 nM. The transport system was clearly distinct from the serotonin transporter. Analysis of the inhibition of dopamine uptake by various monoamine uptake inhibitors showed that the uptake occurred via a transport system that is similar to the neuronal norepinephrine transporter.

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