Effect of iron on early changes in Escherichia coli-induced ascending pyelonephritis in rats

Uptake of d-glucose, l-lysine and l-proline and the kinetic parameters of alkaline phosphatase and γ-glutamyl-transpeptidase were evaluated in renal brush border membrane (BBM) vesicles prepared from control, pyelonephritic and iron-administered pyelonephritic rats. The uptake of d-glucose and amino acids and V_max of both the enzymes studied were found to be altered in pyelonephritic and iron-administered pyelonephritic rats, but changes appeared earlier and more severely in iron-administered infected animals than in other infected animals. These early physiological alterations were accompanied by higher bacterial colonisation in iron-administered rats.     

Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.

Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Phosphoprotein phosphatase activity of the progesterone-induced purple glycoprotein of the porcine uterus.

The iron-containing, progesterone-induced, purple glycoprotein from the pig uterus catalyzes the hydrolysis of phosphate groups from phosphvitin. This enzyme which can be purified in large amounts, has a pH optimum at 4.6 and an apparent K_m of 0.1 mM for bound phosphate. Its V_max with phosphvitin as substrate is about 200, μmoles Pi released/min/μmole enzyme and is at least twenty-fold lower than its activity towards p-nitrophenylphosphate. The enzyme is activated by 2-mercaptoethanol and inhibited by molybdate, fluoride and arsenate. The resemblance of this protein to a number of other acid phosphatases of similar substrate specificity in other organisms, suggests that a broad class of such enzymes might exist in nature.     

Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D₃ was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D_28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca²⁺ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M_R = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D_28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D_28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.     

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.     

Effect of enterotoxigenic Escherichia coli heat stable toxin on intestinal lysosomal enzymes in the suckling rat

A preliminary study on 9 suckling Wistar rats, which received E. coli stable toxin, and on 12 sham-operated controls showed that acid phosphatase, the marker enzyme for lysosome, was significantly increased in the infected group whereas alkaline phosphatase, glucose 6-phosphatase, succinic dehydrogenase, and proteinase, the marker enzymes for brush border, microsome, mitochondria, and the soluble fraction, respectively, remained unaffected. The results suggest that lysosome, the subcellular organelle responsible for intracellular digestion could be modified by E. coli stable toxin. In another set of experiments, where 7 infected suckling rats and 7 sham-operated controls were used, the maximal activities of lysosomal enzymes (released by Triton X-100) were found to be increased in the infected group confirming the results obtained in the preliminary experiment. The values of the ratio between maximal and basal activity (an expression of the degree of retention of enzymes to lysosome) of acid phosphatase and cathepsin D were also significantly increased, indicating that lysosomal membrane may also be stabilized during the infection. The increased activities of lysosomal enzymes and the increased lysosomal membrane stability suggest that intracellular digestion by lysosome could be increased during E. coli stable toxin infection.     

Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.     

Hydrophobic interactions of brush border alkaline phosphatases: the role of phosphatidyl inositol

Tissue-specific (intestinal) and tissue-nonspecific (kidney) rat alkaline phosphatases are released from their respective brush border membranes by different enzymes. To elucidate the mechanism underlying their membrane attachment, we tested the ability of these enzymes to partition into lipid or aqueous phases both before and after treatment with phospholipases and proteases. Interaction with Triton X-114 micelles was eliminated or decreased by treatment of intestinal enzyme with phospholipase A2 or papain, while only phosphatidylinositol (PI)-specific phospholipase C (PIPLC) and subtilisin were effective with the kidney enzyme. Binding to octyl Sepharose for the intestinal enzyme was decreased by phospholipase A2 more than by PIPLC, whereas the reverse was true for the kidney enzyme. Treatment with phospholipases decreased the apparent mass of the phosphatases by 50-80 kDa, presumably due to loss of bound lipid and detergent. PIPLC treatment of the kidney, but not the intestinal enzyme, prevented binding of the phosphatase to phospholipid vesicles. These results show that both enzymes are bound to respective membranes by hydrophobic anchor peptides to which phospholipids are bound. However, their sensitivity to phospholipases is different. The data are consistent with the hypothesis that, in the kidney enzyme, the PI is bound covalently, while with the intestinal enzyme, binding of PI appears to be tight but not covalent.     

Morphological, histochemical, and biochemical studies on the gut of Haemonchus contortus Rud., 1803).

A study has been made on the structure and chemical composition of the gut of Haemonchus contortus (Rud., 1803). The oesophagus has typically a triradiate, cuticle-lined lumen. The intestinal epithelium is provided with a well-developed brush border which contains periodic acid-Schiff-positive mucoproteins. The intestinal epithelium stores glycogen and lipids. It stains diffusely for phospholipids and general proteins and also for terminal-NH2 group. The presence of Fe2+ and Fe3+ containing pigments and activities of acid and alkaline phosphatases, glucose-6-phosphatase, and 5'-nucleotidase have been observed in the intestinal epithelium. Biochemically pH optimum for intestinal acid phosphatase has been found to be 4.8. The brush border shows positive reactions for acid phosphatase and glucose-6-phosphatase, and negative reactions for alkaline phosphatase and 5'-nucleotidase, and negative reactions for alkaline phosphatase and 5'-nucleotidase. The presence of enzymes in the brush border is related to extracellular digestion and absorption of nutrients.     

Some properties of acid and alkaline phosphates from boar sperm plasma membranes

Boar sperm plasma membranes were purified by differential and sucrose density equilibrium centrifugation and were found to yield a single band at a density of 1.14 g/cm3. Both alkaline and acid phosphatase activities were enriched in this fraction. The alkaline phosphatase activity was optimal in 100 mM tris (hydroxymethyl) methylamine (Tris)-NaHCO3 at pH 9.9 with 0.05% Triton X-100 and 1 mM MgCl2. This activity was inhibited by ethylenediaminetetraacetic acid (EDTA), cadmium, zinc or heating at 60 degrees C for 30 min. Also, L-homoarginine caused approximately 70% inhibition and L-phenylalanine or L-leucine caused about 10 to 20% inhibition. Acid phosphatase activity was optimal in 100 mM sodium acetate at pH 5.1 with 0.05% Triton. Sodium dodecyl sulfate, potassium fluoride (KF) or sulfhydryl reagents inhibited the activity, while EDTA or heating at 60 degrees C had no effect. These data for enzymes from boar sperm plasma membranes can be used for future work on the quantitation of the enzymes, distinguishing these two phosphatases from other phosphohydrolases, purification of the enzymes and for comparison to phosphatases in other tissues.     

Salinity-sensitive alkaline phosphatase activity in gills of the blue crab,Callinectes sapidus Rathbun

A levamisole-sensitive (K_i = 0.72 mM) alkaline phosphatase (pH optimum 9.1) and a levamisole-insensitive alkaline phosphatase (pH optimum 7.1) are present in gills of the blue crab Callinectes sapidus. Both enzymes are distinct from ouabain-sensitive ATPase. Specific activity for either phosphatase is greatest in the acinar tissue, which lines the branchial vessels. Histochemical localization of the enzymes confirmed this distribution. Activity of levamisole-sensitive alkaline phosphatase is affected by acclimation salinity. V_max of the levamisole-sensitive alkaline phosphatase is greater in high-salinity crabs than in low-salinity crabs; apparent K_m is not significantly different. The levamisole-sensitive alkaline phosphatase associated with the acinar tissue lining the branchial vessels may modulate the osmoregulatory response in blue crabs.     

The use of octyl beta-D-glucoside as detergent for hog kidney brush border membrane

Octyl beta-D-glucoside was synthetized from alpha-acetobromoglucose with an improved method yielding a very pure product with a sharp melting point (108-109 degrees C) and free of intermediate products as judged by IR and NMR spectra. The yield of the synthesis is 66% when referred to alpha-acetobromoglucose. The potency of this compound as a detergent on hog kidney brush border membranes was compared to the action of Triton X-100. Octyl glucoside preferentially extracts aminopeptidase M and gamma-glutamyltranspeptidase in a concentration-dependent manner. The more deeply imbedded membrane enzyme, alkaline phosphatase, was relatively resistent to the action of octyl glucoside. In contrast, Triton X-100 extracted all membrane proteins to about the same extent. Additionally it was found that octyl glucoside can be removed from membrane extracts by Biobead SM 2. The capacity of the beads is about 170 mg detergent/g of dry Biobead SM 2. Thus octyl glucoside seems to be a useful tool for solubilization and purification of brush border membranes proteins.     

Solubilization of brush borders of hamster small intestine and fractionation of some of the components

About 90% of the protein of hamster intestinal brush borders was solubilised in 0.25% (w/v) sodium dodecyl sulphate without total loss of biological activity. Detergent-polyacrylamide gel electrophoresis of the solubilised protein separated 10–15 bands and partially resolved maltase, lactase, sucrase-maltase, trehalase and alkaline phosphatase activities. The disaccharidases, which were associated with the higher molecular weight proteins, were preferentially solubilised with 0.1%. (w/v) Triton X-100, butanol or papain, whereas Tris and NaI extracted only the lower molecular weight proteins, possible derived from the core filaments.Electrophoresis of brush border proteins metabolically labelled with [¹⁴C] glucosamine suggested that many of the membrane-bound enzymes are glycoproteins. However, chromatography of a papain digest on Sephadex G-200 showed that the sucrase-maltase complex can be separated nearly free of carbohydrate without total loss of activity.The importance of characterizing membrane proteins solubilised by a number of techniques is discussed.     

Delta endotoxin inhibits a phosphatase in midgut epithelial membranes of Heliothis virescens

A vanadate- and delta endotoxin-sensitive phosphatase copurified with plasma membranes and brush border membranes from the midgut epithelium of Heliothis virescens. Phosphatase activity was stimulated under alkaline conditions. Total phosphatase activity was inhibited 60% in the midgut membranes by 360 nM delta endotoxin with a K_i = 76 nM. Brush border membranes were inhibited 75% by 1.47 μM delta endotoxin with a K_i = 64.7 nM. Vanadate (200 μM) completely inhibited the toxinsensitive alkaline phosphatase. A 72 kDa protein was phosphorylated when membranes were incubated with Mg²⁺ and [³²P]orthophosphate, and phosphorylation was inhibited by both vanadate and delta endotoxin, suggesting that destabilization of this phosphoprotein was responsible for phosphatase inhibition.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Studies on the structure of the rabbit kidney brush border.

The effects of salts and non-ionic detergents on renal brush borders have been studied. 2 M sodium chloride, iodide or thiocyanate dissociated up to 40% of the protein from the brush borders, destroying the core filaments and resulting in the formation of membrane vesicles; EDTA had a similar effect on structure but released little protein. Triton X-100 and Nonidet P-40 extracted up to 60% of the protein including the major membrane glycoproteins and the enzymes trehalase, maltase and aminopeptidase (microsomal). Triton exhibited a selective effect on lipids removing phosphatidylserine, phosphatidylethanolamine and sphingomyelin but not the bulk of the phosphatidylcholine or cholesterol. The residual structures after Triton extraction comprised the core filaments associated with vesicles of lipid containing alkaline phosphatase and several other proteins. Treatment of these core-vesicle complexes with 2 M sodium chloride dissociated the filaments, releasing the vesicles which could be recovered as a pellicle on centrifugation. It is suggested that the proteins found in the vesicles might serve to interconnect the core filaments with the lipid bilayer.     

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells

The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K_m value for p-nitrophenyl phosphate, were more sensitive to inhibition by l-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.     

Phosphatases from pollen of Brassica campestris and Lilium regale

Soluble and wall-bound acid phosphatases isolated from rape seed pollen showed similar properties except for the pH optimum curve which was elevated for the cell wall enzyme. About 50 % of the phosphatase activity of washed pollen wall preparations could be solubilized with Triton X-100, compared with only ca 20% for the corresponding preparation from lily pollen. A comparison of the wall-bound acid phosphatase of rape seed and lily pollen showed a marked difference in specificity towards fructose-6-phosphate and glucose-6-phosphate. A Mg²⁺-dependent alkaline pyrophosphatase was obtained from rape seed pollen but this activity could not be detected in cell wall preparations.     

Purification and properties of trehalase from monkey small intestine

Brush border membrane trehalase was purified from monkey small intestine by a procedure which includes solubilisation by Triton X-100, ammonium sulphate fractionation, and chromatography on DE-52 and hydroxyapatite. The purified enzyme had a specific activity of 11 units/mg protein and was purified 140-fold. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis. It had aK ^m value of 17.4 mM for trehalose and a V_max of 1.33 units. Sucrose and Tris acted as competitive inhibitors of the enzyme.