Development of sucrase in the chick small intestine

Development of sucrase in the chick small intestine was studied biochemically and immunologically using antiserum prepared against purified chick intestinal sucrase. Sucrase activity was first detectable at 10 days of incubation and increased with age. After a transient drop at 20 days, the activity rapidly increased to the adult level. Immunodiffusion and polyacrylamide gel electrophoretic studies suggested that the sucrase of the embryonic and hatched chick intestines was identical except for a difference in the content of sialic acids. In immunofluorescence and immunoelectron microscopy, sucrase was found to appear on the luminal surface of epithelial cells at 8-10 days of incubation, soon after the start of morphological differentiation from an undifferentiated thick epithelium to a thin simple epithelium.     

Purification and properties of alkaline phosphatase from the mucosa of rat small intestine

Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, EC 3.1.3.1] was purified from the mucosa of rat small intestine by butanol extraction, ethanol fractionation, gel filtration, with controlled-pore glass-10 and DEAE-cellulose column chromatography. On the gel filtration, the enzyme activity was separated into three peaks; A in the void volume, B and C at lower molecular weight positions. Enzyme A was purified to homogeneity. The activity of enzymes A, B, and C was detected even on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at the position of the protein of enzyme A, which had a molecular weight of 110,000 daltons. Enzymatic properties such as pH optimum, Km value for the substrate, heat inactivation and inhibition by amino acids were the same in all three enzymes. Based on these findings, together with the elution positions on gel filtration, enzyme A was regarded as an aggregate, and enzymes B and C as dimer and monomer molecules, respectively.     

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity.

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.     

Giardia lamblia: expression of alkaline phosphatase activity in infected rat intestine

Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p<0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt-villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p<0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were confirmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These findings indicate differential changes across the crypt-villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine.     

Kinetic characteristics of soluble and brush border alkaline phosphatase and sucrase activities in developing rat intestine: effect of hormones

Suckling rat intestine contains 35 and 65% of the cytosolic and membrane-bound alkaline phosphatase (AP) activities. The corresponding values for sucrase were 20 and 80% respectively. The amount of the soluble enzymes was reduced to 7-11% in adult rat intestine. Administration of cortisone, thyroxine or insulin to suckling animals induced adult type distribution of the enzymes. There were apparent differences in kinetic characteristics of soluble and brush border enzymes, but the kinetic properties of the normally developed and hormone-induced AP and sucrase were essentially similar. This suggested identical nature of these enzymes under these conditions. A biphasic Arrhenius plot was obtained for AP in weaned and hormone injected pups with a break point around 18 degrees C, while the soluble enzyme yielded a monophasic curve (Ea = 8-11 kcal/mole). Arrhenius plot for sucrase was monophasic in the suckling, hormone-injected and adult rat intestine (Ea = 8.3-15.1 kcal/mole). Membrane-bound enzymes were generally labile, while soluble enzyme activities were stable to heat treatment (sucrase at 50 degrees C and AP at 60 degrees C) in various experimental groups.     

Development of the small intestine in the hypophysectomized rat. II. Influence of cortisone, thyroxine, growth hormone, and prolactin.

The intestinal deficiencies caused by hypophysectomy of rats at 6 days of age can be repaired to varying degrees by thyroxine or cortisone but not by growth hormone or prolactin. Administration of daily doses of thyroxine alone from 19–22 days raises duodenal alkaline phosphatase activity to normal levels at 24 days; it has a strong effect on jejunal sucrase and maltase, although these activities remain below those of controls. Thyroxine causes a marked increase in rough endoplasmic reticulum and restores the Golgi complexes to their normal appearance. It also elicits an intensification of periodic acid-Schiff (PAS) stainability of the brush border. Cortisone acetate given from 19 to 22 days elevates sucrase and maltase to normal levels but does not fully restore phosphatase activity. Like thyroxine, cortisone causes intensification of PAS staining of the brush border and also increases rough endoplasmic reticulum. It seems to stimulate Golgi activity, but results in the appearance of a variety of abnormal forms. The defects in Golgi configuration, brush border carbohydrate content, and activity of glycoprotein enzymes that are bound to the brush border may all reflect impaired glycosylation in the hypophyseoprivic state; the results of thyroxine or cortisone administration suggest that both hormones may affect glycosylation but in different ways.     

Physico-chemical properties of alkaline phosphatase from the seal small intestine

Alkaline phosphatase of the Greenland seal was purified to homogeneity, using immobilized concanavalin A. The specific activity of the enzyme is 1200-1500 mu/mg protein. The molecular mass of alkaline phosphatase as determined by electrophoresis performed under non-denaturating conditions is 260 kD, whereas that determined in the presence of beta-mercaptoethanol and SDS is 70 kD, which points to the tetrameric type of the seal alkaline phosphatase molecule. Using the atomic adsorption method, it was demonstrated that the phosphatase molecule contains four zinc atoms. Some physico-chemical parameters of seal alkaline phosphatase (pH-dependence, effects of temperature and cations on the enzyme activity, pI, thermal stability) were determined.     

Effect of simultaneous administration of ibuprofen and ethanol on the alkaline phosphatase activity in the small intestine

Using the interferometric technique the authors studied the effect of simultaneous administration of ibuprofen (Polfa) and ethanol on the activity of alkaline phosphatase in the proximal jejunum. Both ibuprofen and ethanol cause in the digestive tract functional and morphological changes. The used technique of quantitative determination of alkaline phosphatase activity at the site of its primary location made possible an assessment of changes in the activity of the enzyme caused by the administered agents. It was found that after 60 days of the experiment both agents caused a statistically greater changes were observed in the rats receiving both these agents simultaneously. The obtained results suggest the conclusion that simultaneous administration of ibuprofen and ethanol causes in the mucosal gland in the proximal small intestine development of an interaction of the additive.     

Alkaline phosphatase and maltase activity in the embryonic chick intestine in culture. Influence of thyroxine and hydrocortisone.

In strips of duodenum from 14-day chick embryos explanted into defined medium, alkaline phosphatase (ALP) activity accumulated in the tissue at a faster rate than in vivo for about 48 hr, but failed to increase thereafter. The addition of thyroxine (T4) to the medium at 10^?8M or less both enhanced the early accumulation and elicited a very large increase in ALP activity comparable to that normally occurring during the last 2 days in ovo. Activity with phenylphosphate (PhP) was more strongly affected than that with β-glycerophosphate (βGP), so that the high $\text{PhP}\text{β}\text{GP}$ ratio attained in vivo at 18 days was reached after 24 hr in T4-supplemented medium. Hydrocortisone (HC) evoked APL activity only slightly above that in unsupplemented medium and only during the first 48 hr in culture, but it precociously elevated $\text{PhP}\text{β}\text{GP}$ ratio to the normal maximum. In the presence of T4 or HC, maltase activity rose in explanted strips at the same rate as in the intact duodenum, but it lagged in unsupplemented medium. Assay of the medium revealed, however, that under all conditions of culture a large amount of both maltase and ALP activity had been released from the tissue. This effect was especially pronounced in the presence of T4, so that explants and medium together accumulated ALP and maltase equivalent to the high peaks of activity found in the intact duodenum at hatching. With 10^?8M T4, ALP activity began to rise above that of control explants after 8 hr, with accumulation in the medium beginning about 4 hr later. Combining 2 × 10^?6M HC with a range of T4 concentrations produced greater than additive effects, particularly with ALP, but did not lead to enhanced retention of either enzyme in the tissue strips. Prolactin, pentagastrin, and insulin were without effect alone, but the latter inhibited the effects of both T4 and HC.     

Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine.

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.     

Immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment

Summary The purpose of this study was to investigate the effect of colchicine and vinblastine on the localization of alkaline phosphatase (AlPase) in rat duodenum in relation to structural changes. AlPase was localized on the membranes of rough endoplasmic reticulum, Golgi stacks, cytoplasmic vesicles, microvilli, on lateral plasma membranes, and in some lysosomes of the duodenal epithelial cells of rats treated with either lumicolchicine or 0.9% NaCl alone. Microvilli were most intensely stained, and AlPase-positive Golgi stacks were regularly distributed in the supranuclear regions. After colchicine treatment, microvilli were shortened and the staining intensity became weaker, whereas basal as well as lateral plasma membranes showed stronger staining. The AlPase-positive microvilli appeared not only on the luminal surfaces, but also on the baso-lateral plasma membranes and even on the surfaces of characteristic intracytoplasmic cysts. Golgi stacks became smaller and their distribution became less localized, and the staining intensity of the Golgi stacks became weaker. AlPase localization in rats treated with vinblastine was almost identical with that of rats treated with colchicine. Thus, colchicine and vinblastine appeared to have elicited a disorientation of intracellular transport of intestinal AlPase by inhibiting microtubule organization.