Brush Border Membrane Enzymes Research Articles

ALTERATION OF SURFACE MEMBRANE GLYCOPROTEIN SYNTHESIS IN THE SMALL INTESTINE OF RATS WITH NUTRITIONAL IRON DEFICIENCY (NID)

We studied the activity and kinetic parameters of brush border membrane (BBM) enzymes in villus cells and the concentration of the secretory component (SC) of p-IgA in subcellular fractions of crypt cells in 35 day old rats with NID and in controls. In rats with NID, the specific and the total activities of sucrase, maltase, lactase, aminopeptidase and diamine oxidase were decreased from -17 to -66% (p<0.05) compared to the controls. The lower activity of sucrase in the BBM of rats with NID (2.26 times) was associated with much slower enzyme synthesis rate (2.19 times) than in controls. Km of sucrase determined in villus cells was identical in rats with NID and controls but the Vmax was reduced proportionally to the enzymatic activity (1.21 and 1.24 times lower than control values). Electron microscopy of epithelial villus cells from rats with NID revealed a rarefaction of transport vesicles without structural changes of BBM or of the cellular organelles. In villus and crypt cells of rats with NID, SC was reduced to a level about one-half (p<0.05) that of the controls. When SC was measured in subcellular fractions of crypt cells, SC content in each fraction was 2 to 3 times lower in rats with NID than in controls. In conclusion, the decrease in disaccharidases and in SC in the small intestine of NID rats is due to an alteration of the synthesis with reduced transport of these glycoproteins to the BBM.

Effect of External Abdominal Irradiation on Intestinal Morphology and Brush Border Membrane Enzyme and Lipid Composition

Previous studies have shown that external abdominal irradiation is associated with alterations in intestinal morphology and function. The activity of the jejunal brush border membrane (BBM) enzyme markers sucrase (S) and alkaline phosphate (AP) were not altered by 600 rad irradiation in the rat. In contrast, ileal BBM, AP, and AP/S were increased 3, 7/8, and 28 days postirradiation. The total lipid composition of the jejunal BBM was lower than in control animals only at 3 days postirradiation; this was due to a decrease in the total free fatty acid content. In addition to a lower total free fatty acid content, the ileal BBM contained an increased amount of total phospholipid (PL) which resulted in an increased phospholipid/cholesterol ratio at 3 days following irradiation. Variations in the BBM phospholipid composition occurred in both jejunum and ileum. In the jejunal BBM, the phospholipid composition changes did not alter the choline or amine phospholipid content; therefore, the choline/amine phospholipid ratio was unaffected by irradiation at 600 rad. In the ileal BBM, the phosphatidyl ethanolamine was increased at 3, 7/8, 14, and 28 days following irradiation. The choline/amine phospholipid ratio was not altered in the ileal BBM due to concomitant increases in lecithin content. Jejunal villus height, villus surface area, and the number of cells per villus were decreased at 3 days postirradiation, but increased by day 7/8 and 14 postirradiation to levels much higher than observed in control jejunal villi. The mucosal surface area was decreased at 3 and 7/8 days following irradiation but returned to control values by Day 14. Jejunal microvillus morphology was unaffected by irradiation. Few significant changes were observed in ileal villus morphology following irradiation at 600 rad. Ileal villus height, villus surface area, and mucosal surface area did not change, but the number of cells per villus initially decreased at 3 days and then increased beyond control values at 7/8 and 14 days postirradiation. Ileal microvillus height was significantly decreased only at 7 days postirradiation, while the number of microvilli per micron was increased only at 3 days postirradiation. This study suggests that changes in intestinal morphology and brush border composition may contribute to the altered passive permeation toward lipids which has been reported following abdominal radiation.

The development of the small intestine.

The remarkable degree of coordination between the development of various aspects of gastrointestinal function suggests that the process may be triggered by a single or a few central mechanisms, such as weaning and (or) hormones. Precocious development of enzyme and transport function can be induced by exogenous thyroxine and corticosteroids, while thyroidectomy and adrenalectomy abolish the normal pattern of postnatal development. These hormones may have a primary or a permissive role. Activation of the dormant hormonal mechanism could be controlled by a genetically coded biologic clock, such as chronologic age, or by a biological signal such as body size and oral intake. Generally speaking, shortly after birth, there are increases in the intestinal mucosal surface area, brush border membrane enzymes, and carrier-mediated transport. These adaptive changes occur as a result of the genetic endowment of the animal, but may be modified by environmental factors, particularly nutrient intake.

Resection of rabbit ileum: effect on brush border membrane enzyme markers and lipids

Alterations in transport function have been described 6 weeks after surgical resection of 50% of the distal small intestine. Previous studies demonstrated a modest increase in the jejunal uptake of medium chain length fatty acids following resection, while the uptake of many other lipids (cholesterol, bile acids, fatty alcohols, short and long chain length fatty acids) appears to be unaffected. Marked changes in the kinetic constants for the carrier-mediated uptake of four sugars and leucine were observed following resection, but the changes in transport were not associated with changes in the mucosal surface area. This study was undertaken to examine the possible adaptive mechanisms that occur with ileal resection in the rabbit. A 29% increase in the wet weight of jejunal mucosal scrapings and a 53% increase in jejunal brush border membrane (BBM) protein was observed following resection. The jejunal BBM sucrase (S) was unchanged following ileal resection, but alkaline phosphatase (AP) total activities were increased in the resected rabbits. This resulted in a 45% increase in the ratio of AP/S with resection. The lipid composition (total free fatty acids, total bile acids, total cholesterol, total phospholipids, individual phospholipids, and the ratio of total phospholipids/total cholesterol) of BBM was similar in control and resected rabbits. This suggests that quantitative rather than qualitative changes in the membrane composition may be responsible for the transport changes observed in resected animals.

NAD-glycohydrolase in renal brush border membranes

NAD is hydrolyzed during incubation with isolated renal brush border membranes (BBM). The specific enzymatic mechanisms have not been identified apart from the activity of ADP-ribosyltransferase, which accounts for a very small proportion of the total hydrolysis. In the present study, an NAD-glycohydrolase (NGH) was identified in the renal BBM using the cyanide-addition assay to monitor hydrolysis of NAD at the nicotinamide-ribose bond. The production of nicotinamide and ADP-ribose, the expected reaction products, was determined by thin-layer chromatography. The NGH was enriched ninefold in the BBM fraction and accounted for 36% of the total rate of NAD hydrolysis by BBM enzymes at pH 7.4. Assay of NGH in sealed BBM vesicles subjected to osmotic shock indicated that about 23% of the NGH is exposed on the cytoplasmic surface of the BBM. The enzyme was inhibited by nicotinamide in vitro and also when the nicotinamide was administered in vivo, suggesting, indirectly, that the enzyme may play a role in mediating the effects of nicotinamide on BBM phosphate transport.

Intestinal brush border membrane marker enzymes, lipid composition and villus morphology: Effect of fasting and diabetes mellitus in rats

1. 1. Diabetes mellitus is associated with enhanced passive intestinal uptake of cholesterol and fatty acids. 2. 2. In order to determine the basis for these changes in intestinal permeability, the jejunal morphology and the lipid content of purified brush border membranes (BBM) were measured in fasted and fed control (C) and streptozotocin diabetic (DM) rats. 3. 3. There was no difference between C and DM in BBM sucrase or alkaline phosphatase; fasting had no effect on BBM enzymes in C, but in DM fasting was associated with increased sucrase activity per length of jejunum. 4. 4. In C fasting was associated with higher levels of BBM total phospholipid, lecithin, choline and amine phospholipids, whereas fasting in DM was associated with higher BBM cholesterol and lower free fatty acids. 5. 5. In the fasting DM, there was a greater villus and mucosal surface area than in the fasting C. 6. 6. A previous study demonstrated that with fasting in DM versus C, cholesterol uptake was unchanged, but when animals were fed, cholesterol and fatty acid uptake were greater into the jejunum of fed DM as compared with fed C. 7. 7. In the BBM of fed DM as compared with C, there was a significant increase in total phospholipid, lecithin, phosphatidyl ethanolamine, choline and amine phospholipids, and phospholipid/cholesterol ratio. 8. 8. Thus, (1) fasting is associated with changes in intestinal morphology, BBM lipids; (2) the effect of fasting is different in DM and C; (3) the enhanced uptake of lipids into the jejunum of fed diabetic rats is not due to changes in villus morphology, but may be due to alterations in the BBM phospholipids.

Aminoglycoside-induced functional and biochemical defects in the renal cortex

The first site of aminoglycoside-cell interaction occurs at the plasma membrane of renal proximal tubular cells which have been shown to selectively transport and accumulate these drugs. Depression of apical membrane transport of organic base, low-molecular-weight protein, and glucose, together with loss of brush border membrane enzymes and phospholipids in the urine which results in altered phospholipid composition of this membrane, occurs early in the course of aminoglycoside administration. Less well appreciated are the alterations which occur at the basolateral membrane. These include decreased transport of organic bases, Ca 2+, Na 2+, and K +; increased organic acid transport; decreased activity of Na +-K + ATPase and adenylate cyclase; decreased calcium content; and altered phospholipid composition. Many of these changes are evident within 90 min of a single injection of drug. Lysosomal dysfunction is manifested by the accumulation of phospholipids in the form of myeloid bodies consequent to the inhibition of lysosomal phospholipases by aminoglycosides. Labilization of lysosomes in vivo has been postulated to be a mechanism of cell injury. Mitochondrial dysfunction attributed to aminoglycosides includes impaired respiration, inhibition of Mg 2+ binding, inhibition of Ca 2+ uptake, increased permeability to monovalent cations, decreased ammoniagenesis, and decreased gluconeogenesis. However, it remains unclear how the drug gains access to mitochondria in vivo in order to initiate the functional derangements. It is evident that aminoglycosides cause multiple metabolic derangements at multiple sites within renal proximal tubular cells. At present the available evidence does not identify which, if any, of these drug effects is responsible for initiating the injury cascade. The strong possibility exists that aminoglycoside nephrotoxicity reflects the net impact of multiple minor metabolic derangements which individually are of little significance but when added together seriously compromise the cell's ability to maintain its structural and functional integrity.

De novo synthesis of brush border membrane enzymes during intestinal perfusion with bile salt in the rat.

Jejunal perfusion in the rat with Ringer solution containing 10 mmol/l taurocholate removes considerable quantities of protein and brush border membrane enzymes from the intestinal epithelium. The duration of the experiments was 7.5 h. One group of animals was given 200 micrograms cycloheximide per 100 g body weight intramuscularly 1 h before start of the perfusion. Serial estimations of protein and of four brush border membrane enzymes (alanine aminopeptidase, alkaline phosphatase, gamma-glutamyl transferase, and enteropeptidase) were done in the perfusate. The results provide evidence that during the experiments an increasing proportion of the enzymes stems from de novo synthesis. This is consistent with the concept that after loss of 10-30 per cent of enzyme the molecules are replaced by newly synthesized material, provided that the energy metabolism of the mucosa cells remains intact.

Biochemische und morphologische untersuchungen zum einfluß von gallensäuren auf das epithel im jejunum der ratte

The influence of bile salts on the mucosal surface of rat jejunum was tested with an in vivo technique of segmental perfusion. Sodium taurocholate and chenodesoxycholate were applied in a concentration of 3 mmol/l. The release of 5 brush border membrane enzymes, 5 cytosolic, 1 mitochondrial, and 2 lysosomal enzymes during a perfusion time of 150 min as well as morphological alterations after bile salt treatment were investigated. Among the membrane enzymes, due to their superficial localization, the solubilization of enteropeptidase and alpha-1,4-glucosidase was highest both in the control perfusion and in the presence of bile salts. At the same time, cytoplasmic enzyme activities were liberated extensively whereas lysosomal and mitochondrial enzymes were scarcely detectable. This disproves any serious injury of the enterocytes. Electronmicroscopic results supported this suggestion. After administration of taurocholate (in physiological concentration), only an occasional diminution of the glycocalyx was observed and even chenodeoxycholate (in an unphysiological concentration) caused only negligible destructions of intestinal brush borders. Investigations with ruthenium red to contrast the glycocalyx showed a partially unchanged structure. Microvesiculation from the microvilli was observed in many electron microscopic photographs. That is a possibility for the release of membrane-bound and cytosolic enzymes without destruction of enterocytes.

Comparative study of some intestinal brush border membrane enzymes during perinatal development of the mouse

1. 1. Intestinal brush border membrane (bbm) fractions have been isolated from fetal and neonatal mice. 2. 2. The existence of discordant developmental patterns of intestinal enzymatic activity derived from total homogenate and bbm fraction was confirmed. 3. 3. It originates chiefly from two phenomena: (a) variations in the state of purity of brush border fractions, and (b) loss of brush border membrane enzyme activities in supernatant that increases with age. 4. 4. The phenomenon of solubility for glucoamylase and alkaline phosphatase is already present two days before birth.

NAD+-dependent ADP-ribosyltransferase in renal brush-border membranes.

Oxidized nicotinamide adenine dinucleotide (NAD+) in cytosol may interact with renal brush-border membranes (BBM) and inhibit BBM phosphate transport. The possible mechanism of interaction was investigated in the present study. Incubation of BBM with [adenine-3H]NAD+ led to acid-stable binding of 3H to the BBM, in contrast there was no binding of 14C when [carbonyl-14C]NAD+ was used. The data are consistent with an ADP-ribosylation mechanism involving transfer of ADP-ribose from NAD+ to BBM. This was confirmed by using [adenylate-32P]NAD+ and by the release of bound 32P in the form of 5'-[32P]AMP when the BBM were treated with snake venom phosphodiesterase. After gradient centrifugation of BBM the ADP-ribosyltransferase was recovered at the same density as known BBM enzymes, indicating that ADP-ribosyltransferase is an intrinsic BBM component and not a contaminant. These findings indicate that cytosolic NAD+ may be used for ADP-ribosylation of BBM proteins and that this may be a mechanism for regulating the BBM phosphate transport system.

Release of enzymes from rat jejunal mucosa by bile salts.

The technique of segmental perfusion was applied in vivo to study the release of enzymes from the mucosa cells under the influence of bile salts. Five brush border membrane enzymes, five cytosolic, one mitochondrial and two lysosomal enzymes were measured. Spontaneous release (Ringer solution), due to their superficial localization, was greatest for enteropeptidase and alpha-glucosidase. 3 and 10 mmol/l sodium taurocholate, and 0.5 and 3.0 mmol/l chenodeoxycholate were used. Surprisingly, the majority of cytosolic enzymes was released to a greater extent than membrane enzymes. On the other hand, lysosomal or mitochondrial enzymes were low or absent in the perfusion medium, a finding that excludes a serious injury to the mucosa cells. It must be inferred, therefore, that cytosolic enzymes are regularly externalized during the digestive process, the glycocalyx perhaps being a common matrix for pancreatic, membrane-associated and cytosolic enzymes.

Enzymuria: a parameter for the assessment of potential nephrotoxicity of cephalosporins.

Ten informed healthy volunteers with normal renal function received 2 X 3.0 g ceftazidime on three consecutive days. The combination of the same dose of ceftazidime together with tobramycin (3 mg/kg/d) was administered four weeks later to the same group of volunteers. In the first trial with ceftazidime alone no changes in the elimination of brush-border membrane enzyme AAP were found. In contrast, the administration of ceftazidime together with tobramycin produced the expected response to tobramycin with the cumulative pattern of enzyme elimination into urine. Functional parameters remained unchanged.

Zur histochemischen Erfassung einer β-Hydroxybutyrat-Dehydrogenase im Bürstensaum der Hauptstückepithelien der Rattenniere

NAD-beta-hydroxybutyrate dehydrogenase activity was investigated in the kidneys of male and female adult Wistar rats using membrane incubating technique. In this connection the enzyme activity presents an other topochemical pattern as in comparison with conventional incubating technique. In the kidney cortex an additional reaction was demonstrated in the convoluted portion of proximal tubules. In connection with the results of biochemical investigations the soluble enzyme is characterized as a nonmitochondrial beta-hydroxybutyrate dehydrogenase of proximal kidney tubules (brush border membrane enzyme?).

Metabolism of NAD by isolated rat renal brush border membranes.

NAD and NADH are metabolized to adenosine, ribosyl nicotinamide and Pi by isolated rat renal brush border membranes (BBM). The similar distribution like the BBM marker enzyme alkaline phosphatase indicates that nucleotide pyrophosphatase and 5-nucleotidase are BBM enzymes.

Turnover of mouse intestinal brush border membrane proteins and enzymes in organ culture. A direct evaluation from studies on the evolution of enzyme activities during the culture.

The turnover of mouse intestinal brush border membrane enzymes has been studied by kinetic analysis of the evolution of enzyme activities during organ culture. By comparing the results obtained in these studies with the predictions from a mathematical model of enzyme synthesis and degradation in organ cultures, it has been possible to reach the following conclusions: (1) There is no degradation of brush border membrane enzymes during culture and the rate of synthesis of each enzyme is directly measurable from the kinetics of total enzyme accumulation (tissue + media). (2) Brush border membrane enzymes are released in culture media by two complementary processes. The first one involves a differential solubilization of enzymes but its exact nature cannot be exactly stated. The second one involves a microvesiculation of brush border membranes, the importance of which in vivo is seen in the possible conciliation between urinary membrane synthesis and heterogeneous turnover of membrane components.

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles

The temperature dependence of sodium-dependent and sodium-independent d-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent d-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15°C. The sodium-independent uptake of d-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35–5°C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4°C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.

Nephrotoxicity of cefotiam (CGP 14221/E) in rats and rabbits.

Cefotiam (CGP 14221/E; SCE 963), a semisynthetic cephalosporin, was administered as a single dose by i.v. injection to rats l(up to 1.8 g/kg body-weight) and rabbits (up to 1.7 g/kg body-weight). Cephaloridine served as positive control (1.0 and 0.75 g/kg in rats; 0.3 and 0.28 g/kg in rabbits). The animals were sacrificed 24 h after injection and the kidneys preserved for routine histology and enzyme histochemistry (alkaline phosphatase, aminopeptidase, succinate dehydrogenase, esterase). Serum samples (Na+, K+, Cl-, urea, creatinine, LDH, alkaline phosphatase) and 24-h urine (Na+, K+, Cl-, urea, creatinine, protein, LDH, aminopeptidase) were analysed before and 24 h after injection. Minimal, irregularly scattered, degenerative changes in the proximal tubules which were not dose-dependent in degree were observed in rat kidneys following cefotiam injection. A slight dose-dependent degeneration in up to 50% of proximal tubular cells with loss of brush-border membrane enzyme activity was observed in rabbit kidneys. In both animal species the ability to concentrate urine was retained and urea and creatinine serum levels hardly affected. Following cefotiam injection a dose-dependent 4-fold excretion of urinary protein but not of LDH was observed in rabbits only. By contrast, cephaloridine caused extensive degeneration and necrosis in up to 90% of proximal tubular cells in both rats and rabbits, which was accompanied with formation of enzymically active hyaline casts, loss of urine-concentrating capacity of the kidney, elevated serum levels of urea and creatinine and an increased urinary excretion of LDH (60-fold in rats, 20-fold in rabbits) and protein (3-fold in rats, 10-fold in rabbits). Histochemistry and electron microscopy of rabbit kidneys suggested a loss of microvilli from proximal tubule cells by endocytosis and thus degeneration following injection of large doses of cefotiam, whereas cell disruption and necrosis prevailed after cephaloridine. The action of cefotiam on the proximal tubule cells is, therefore, not only quantitatively but possibly also qualitatively different from that of cephaloridine. Semiquantitative evaluation of tubular injuries in alkaline phosphatase-stained kidney sections and measurements of LDH and protein content in 24-h urine samples were advantageous in determining the quantity and the quality of nephrotoxic effects.

Quantification of nephrotoxicity in rabbits by automated morphometry of alkaline phosphatase stained kidney sections.

Male and female rabbits were injected intravenously with a single dose of either cefroxadine or cefsulodin or cephaloridine. Quantitative determinations of the activity of two brush border membrane enzymes, aminopeptidase and alkaline phosphatase, were made in homogenates of cortical kidney tissue, in the urine and morphometrically in proximal tubules of cryostat sections. Morphometry was done by classification and enumeration of proximal tubule sections with the same level of enzyme reaction product using a microscopic television analysis system. By comparison with the control values, no changes were detectable 24 h after the injection of up to 1.2 g cefroxadine or cefsulodin per kg body weight. By contrast, after 300 mg/kg cephaloridine, the concentrations of the two enzymes were decreased in a large number of proximal tubules, i.e. the brush border membranes, and concomitantly cell degeneration and necrosis took place. Alkaline phosphatase activity in sections and tissue homogenates was reduced to a greater extent than aminopeptidase activity. A corresponding, significant increase in enzymic activity in the urine was only demonstrable in respect of aminopeptidase. The classification of proximal tubules in sections by television analysis on the basis of alkaline phosphatase reaction product concentration appears to be a reliable measure for detecting and quantifying toxic effects on proximal tubules of kidney.

The interorgan metabolism of glutathione

1. 1. Glutathione is the primary low molecular weight cellular thiol and a major reserve of cysteine. Glutathione is synthesized in the cytoplasm, where it participates in a variety of reactions as an electron donor or a nucleophilic cofactor. 2. 2. However, it now appears that the initial step in glutathione turnover is its release from the cell. The released glutathione is transported by the blood plasma to the kidney where it is efficiently extracted and degraded to its constituent amino acids. 3. 3. The initial reaction in the renal degradation of glutathione is catalyzed by y-glutamyltranspeptidase. which is localized on the external surface of the brush border membrane. 4. 4. Within the lumen of the renal proximal tubule, the γ-glutamyltranspeptidase apparently catalyzes a hydrolytic rather than a transpeptidation reaction. 5. 5. The resulting eysteinylglycine is hydrolyzed by two extracellular brush border membrane enzymes. aminopeptidase M and a recently identified peptidase. 6. 6. Reabsorption of the amino acids and redistribution of cysteine for protein synthesis or glutathione resynthesis completes the interorgan metabolism of glutathione.