Integration of alkaline phosphatase in the intestinal brush border membrane

Alkaline phosphatase has been solubilized from porcine intestinal mucosa by two different methods: treatment of the mucosa by Emulphogen BC 720 and papain hydrolysis of enterocyte brush border membrane vesicles. Two different enzyme forms have been obtained by these methods.The two enzyme forms (‘detergent form’ and ‘papain form’) have been purified to homogeneity by similar techniques and exhibit closely related molecular characteristics. However, the detergent form displays a hydrophobic behaviour and aggregates in media free of detergent. The two forms can be differentiated by their electrophoretic mobility on polyacrylamide gel in the absence of sodium dodecyl sulphate.By electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate, it has been shown that the detergent and papain forms of alkaline phophatase are dimers consisting of two apparently identical subunits whose molecular weights are 64 000 and 61 000, respectively. The difference between these molecular weights has been attributed to the existence of a hydrophobic region in the detergent form which is present on each subunit.     

Ca2+ inhibition of brush border alkaline phosphatase activity in Hymenolepis diminuta

The brush border membrane of Hymenolepis diminuta contains several Ca2+-dependent enzymes. Following our isolation of a Ca2+-dependent modulator protein we examined the kinetic properties of the brush border marker alkaline phosphatase from fractionated and crude tegument. We show that this enzyme is inhibited by Ca2+ concentrations approaching those in the calcareous corpuscles of H. diminuta.     

Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa

In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions.     

Characterization of brush border membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine

This study was conducted to characterize enterocyte apical membrane-bound alkaline phosphatase activity in different segments of the porcine small intestine. Duodenal, jejunal, and distal ileal segments were isolated from three 26-kg pigs and enterocyte brush border membrane, enriched between 19- and 24-fold in sucrase specific activity, was prepared by Mg(2+) precipitation and differential centrifugation. With P-nitrophenyl phosphate as substrate, the optimum pH for porcine brush border membrane-bound alkaline phosphatase activity was defined to be 10.5 for all three segments. At the optimal pH, the kinetics of membrane-bound alkaline phosphatase were determined for the three intestinal segments. The affinity of this enzyme (K(m), mM) in the jejunum (0.64 +/- 0.07) was four times greater than that in the duodenum (2.75 +/- 0.59) and the distal ileum (2.71 +/- 1.14). These results indicate that different isomers of membrane-bound alkaline phosphatase might have been expressed in different segments of porcine small intestine. The maximal specific activity (V(max), micromol/mg protein . min) of this enzyme was highest in the duodenal (7.74 +/- 0.95), intermediate in the jejunal (4.31 +/- 0.18), and lowest in the distal ileal (3.53 +/- 0.84) brush border membrane. Therefore, the maximal specific activity of brush border membrane-bound alkaline phosphatase along the intestinal longitudinal axis in growing pigs decreases from the duodenum toward the distal ileum.     

Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.     

Participation of alkaline phosphatase in the active transport of phosphates in brush border membrane vesicles

This paper reports the separation and preliminary characterization of the products formed by the reaction of the antitumor compound cis-Pt(NH₃)₂Cl₂ with DNA. Electrophoresis of the acid hydrolysed platinum-DNA complex gave a profile of platinum concentration which contained 5 peaks whose relative intensities varied with the amount of cis-Pt(NH₃)₂Cl₂ fixed on the DNA. Similar analysis of the products formed between DNA and trans-Pt(NH₃)₂Cl₂ or [Pt(dien)Cl]Cl, which are not active antitumor agents, indicated that these compounds bound to DNA in a different manner than cis-Pt(NH₃)₂Cl₂. DNA isolated from Escherichia coli which had been treated with cis-Pt(NH₃)₂Cl₂ or [Pt(dien)Cl]Cl did not give the same electrophoresis profiles as the corresponding platinum-DNA complexes formed in vitro.     

Effect of harmaline on rat intestinal brush border sucrase activity

The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing K(m) by 30%, while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Abstract

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium

The alkaline phosphatases present on isolated brush border and basal lateral membranes of rat duodenal epitheilum were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6–9.8, similar substrate k_m's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin or papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000–150,000, and both enzymes show an R_f value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The V_max of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 μmoles/mg-h as opposed to 14 μmoles/mg-h. The differences in V_max are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption

Absorption of dietary fat in the small intestine is accompanied by a rise of intestinal alkaline phosphatase (IAP) in the serum and of secretion of IAP-containing surfactant-like particles from the enterocytes. In the present work, fat absorption was studied in organ cultured mouse intestinal explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A lipid analysis revealed that fat absorption caused a marked increase in the microvillar membrane contents of free fatty acids. In conclusion, fat absorption rapidly induces a transient clathrin-dependent endocytosis via coated pits from the enterocyte brush border. The process selectively internalizes IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles.     

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.     

Translocation of intestinal alkaline phosphatase in streptozotocin-induced diabetic rats

1. We determined the organ of origin and possible mechanism of translocation into the circulation of alkaline phosphatase (ALPase) in the diabetic rat. 2. Experimental diabetes was induced by injection of streptozotocin, resulting in a 8.2-fold elevation in serum ALPase activity. In this case, the major ALPase isozyme detected in serum was intestinal ALPase. 3. In in vitro experimental systems, ALPase was readily released from the duodenal plasma membrane by bacterial phosphatidylinositol-specific-phospholipase C (PI-PLase C) but little if any was released from the ileal membrane. 4. Serum and ileal ALPases were identical in terms of molecular size, whereas duodenal ALPase clearly differed from the serum enzyme. 5. Based on an investigation of the sugar moiety, more of the fraction having higher concanavalin A affinity was found in serum ALPase than with in the case of either of the intestinal ALPases. Serum and intestinal ALPases also differed slightly regarding isoelectric points. 6. Consequently, these data suggest that the serum ALPase of the diabetic rat is derived from ileal ALPase, and it is unlikely that the appearance of ALPase in the circulation is simply the result of solubilization by the action of PI-PLase C or phospholipase D.     

Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma

The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V(max)) and increase in affinity constant (K(m)) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.     

Role of the intestinal brush border in the absorption of cholesterol in rats

1. Short-term incubation of the everted intestinal sacs of rats in media containing cholesterol oleate or cholesterol plus oleic acid resulted in rapid hydrolysis, but no synthesis, of the sterol ester. 2. On separation of the brush border from the rest of the mucosal cell, almost all of the hydrolytic activity and appreciable amounts of the synthetic activity of the whole cell were found to be present in the brush-border fraction. 3. The isolated brush-border fraction contained considerable amounts of cholesterol, which was always present in the unesterified state; the rest of the cell contained about an equal amount of unesterified cholesterol, but, in addition, small but definite amounts of the esterified sterol were also found in this fraction. 4. On feeding rats with [4-(14)C]cholesterol, which was diluted with 3mg. of cholesterol, it was found that the brush border very rapidly took up the fed sterol without changing its net content of cholesterol. No traces of radioactive cholesterol ester could ever be detected in the isolated brush border after feeding with (14)C-labelled esterified or unesterified cholesterol. 5. The appearance of the labelled sterol was quite rapid in the rest of the cell also, where small proportions were found in the esterified state. 6. Therefore the sequence of events in the absorption of cholesterol appears to be: the dietary cholesterol esters are hydrolysed by the cholesterol ester hydrolase of pancreas or of the mucosal brush border or both, after which the brush border rapidly absorbs the de-esterified sterol and transfers it into the mucosal cell, by a mechanism of displacement, where it is slowly re-esterified for transport through the lymph.     

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [³H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3′-sulfonate (Woodward’s Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H⁺ exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H⁺ antiport mechanism.