Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis

The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. gamma-Glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.     

In vitro study of the intestinal brush border enzyme activities in developing anuran amphibian: effects of thyroxine, cortisol, and insulin

The effects of several hormones on intestinal brush border membrane enzymatic activities have been investigated in intestinal explants taken from the amphibian midwife toad at different developmental stages. Explants were treated for at least 2 days with thyroxine (0.1 microgram/ml of culture medium) or for 2 days with cortisol (25 micrograms/ml) or insulin (6 mU/ml). The hydrolases examined were maltase, trehalase, glucoamylase, and alkaline phosphatase. In the explants from tadpoles in prometamorphosis, thyroxine had no effect on hydrolase activities; cortisol increased the activity of only glucoamylase, and insulin increased activity of maltase, glucoamylase, and alkaline phosphatase. When the explants were taken from tadpoles at the beginning of climax, cortisol and insulin generally stimulated the enzyme activities studied. When taken from tadpoles at the end of climax, at the moment when the embryonic cells under the degenerating epithelium divide, cortisol and insulin had little effect on these activities. When the animals terminate their metamorphosis, the intestinal epithelium of the explants is totally newly formed (secondary epithelium). At this time, cortisol stimulated the activities of maltase, glucoamylase, and alkaline phosphatase, while insulin decreased the activities of maltase and glucoamylase.     

Organ culture of adult mouse intestine

Summary Explants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase is measured during the first 24h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.This work was supported by research grants from the Medical Research Council of Canada (J.S.H.) (D.M.)Mr. Chabot is a recipient of a studentship from the Medical Research Council of CanadaD. Ménard, Ph.D. is chercheur-boursier from the CRSQ     

Comparative study of the effect of hydrocortisone and thyroxine on suckling mouse small intestine in organ culture

The influence of hydrocortisone (10(-8)--10(-5) M) and thyroxine (10 (-9)--10(-6) M) on intestinal epithelial cell differentiation and proliferation have been studied using explants of suckling mouse jejunum maintained in serum-free organ culture. Hydrocortisone induced the appearance of sucrase activity and increased trehalase, glucoamylase, lactase and alkaline phosphatase activities. Thyroxine was completely ineffective at all the concentrations used. None of these hormones affected the mitotic activity or the 3H-thymidine incorporation into DNA. These results demonstrate that hydrocortisone but not thyroxine acts directly on intestinal brush border membrane differentiation and that both hormones do not influence the proliferation of the epithelial cells during postnatal development.     

Histochemical studies on the effect of thyroid hormone on alkaline and acid phosphatase activities in liver of fish and amphibia.

The Lata fishes (Ophicephalus punctatus) showed increased alkaline and acid phosphatase activities in liver after immersion for 15-30 days in thyroxine-containing medium (0.025 mug/ml). A single injection of thyroxine (1-2 mug/g of body weight) caused increased acid phosphatase activity in liver of Lata fish in comparison to the controls on the 5th day after experiment but the alkaline phosphatase activity remained unchanged. Both alkaline and acid phosphatases showed increased activities in liver of Lata fishes treated with a single injection of 4 mug of thyroxine per g of body weight on the 5th day. Immersion of Lata fishes in thiourea solution (1 mg/ml) for 15 days did not show any alteration in alkaline or acid phosphatase activities but these enzyme activities decreased after 30 days' immersion in thiourea solution in comparison to the controls. A seasonal variation of alkaline and acid phosphatase activities was observed in liver of Lata fishes. More alkaline phosphatase activity was found in liver of summer fishes than in winter fishes. The winter fishes showed more acid phosphatase activity than the summer fishes. Three consecutive injections of thyroxine (0.1 mug/g of body weight) to toads (Bufo melanostictus) caused increased alkaline and acid phosphatase activities in liver on the 5th day of the experiment, in comparison to the controls.     

Metabolic variants of intestinal alkaline phosphatase in relation to fat absorption: in situ demonstration with the organ-specific inhibitors l-phenylalanine and l-homoarginine

Summary The localization of rat intestinal alkaline phosphatase has been studied in relation to fat absorption. The observations support a theory of conversion, within the intestinal mucosa, of intestinal type to liver type alkaline phosphatase when the criterion of differential sensitivity to two amino acid inhibitors, l-phenylalanine, and l-homoarginine, is applied.Following a three hour in vivo exposure to mixtures of oleic acid, sodium taurocholate, and lauric acid, the epithelium becomes depleted of its l-phenylalanine-sensitive, intestinal type alkaline phosphatase. At the same time, enriched activity is seen in the lamina propria; this activity is both particulate and diffuse, and is present both in the connective tissue matrix and in cells, including macrophages, eosinophils and lymphocytes. Most of this enzyme is inhibited by l-homoarginine, a property characteristic of liver type alkaline phosphatase.The localization of enzyme-positive particles 0.5 to 1.0 in diameter in both epithelium and lamina propria appears identical to that of particulate fat. A physical association between transport of absorbed fat and metabolic conversion of intestinal type alkaline phosphatase is postulated.This work was aided in part by grants-in-aid [CA-3332-01, K6-CA-18,453] from the National Cancer Institute, National Institutes of Health, U.S.P.H.S. and the John A. Hartford Foundation, Inc.Pre-doctoral trainee, U.S.P.H.S. grant GM01451.Holder of a Juan Marsh Foundation Fellowship, Lemuel Shattuck Hospital.     

Organ culture of suckling rat intestine: comparative study of various hormones on brush border enzymes

Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10(-6) M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10(-8) M) and dbcAMP (10(-3) M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity.     

Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration.

A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...     

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.     

Histochemistry of the development of the digestive system of Siberian sturgeon during early ontogeny

At hatching, the yolk-sac matrix of Siberian sturgeon Acipenser baeri contained neutral glycoconjugates, glycogen, proteins rich in arginine, lysine, tyrosine, cysteine and cystine, glycoproteins containing mannose (Man) and/or glucose (Glc), N -acetyl-D-galactosamine (GalNAc), L-fucose (Fuc), sialic acid and/or N -acetyl-D-glucosamine (GlcNAc) residues, as well as neutral and acidic lipids. Buccopharyngeal and anterior oesophageal goblet cellls produced a combination of neutral and acid sialoglycoproteins, while those from the posterior oesophagus secreted only neutral glycoproteins; both types of secretions contained tryptophan and -S-S- groups and were unreactive to lectin techniques. Most intestinal goblet cells secreted mainly carboxylated and sulphated sialoglycoproteins with some rests of neutral glycoconjugates, while few of them produced only acid or neutral glycoproteins. Intestinal glycoproteins were rich in GalNAc, GlcNAc and sialic acid residues. Close relationships between digestive enzymes and morphological development of digestive organs were observed. Histochemistry of enzymes revealed that just after hatching, alkaline and acid phosphatase, ATP -ase and non-specific esterase activities were detected in the yolk sac. From the onset of exogenous feeding to the juvenile stage (30 days post-hatch), an enhancement of enzymatic activities was observed, as alkaline and acid phosphatase, ATP -ase, aminopeptidase M and nonspecific esterase sharply increased. However, lipase activity decreased in the liver and brush border of enterocytes by 13–14 days post-hatch. Two types of lipase were detected in the alimentary canal, a non-pancreatic lipase that was secreted in the cardiac stomach by gastric glands, and a pancreatic lipase, which activity was mainly detected in the brush border of the intestinal epithelium.     

Apical plasma membrane-bound enzymes of rabbit uterine epithelium

Summary In order to monitor changes in the apical cell membrane of rabbit uterine epithelium which are postulated to be a precondition for trophoblast attachment, the marker enzymes: alkaline phosphatase, aminopeptidase M, -glutamyl transferase and dipeptidyl peptidase IV were investigated during the periimplantation phase. Endometrium of early pregnancy (implantation chamber, interblastocyst endometrium; 5–8 days post coitum, d p.c.) was compared with specimens obtained at hCG-induced pseudopregnancy (p. hCG) to distinguish between membrane changes regulated by maternal plasma steroid hormones and such which might be induced locally by blastocyst-derived signals.All enzymes tested showed their main activity at 5 d p.c./p. hCG. The weakest reaction in this series of stages was generally found at 8 d p.c. (interblastocyst segments) or at 8 d p. hCG. In contrast to the rest of the epithelium, the implantation chamber retained high activity of dipeptidyl peptidase IV, and the activity of alkaline phosphatase even raised here again at 7 and 8 d p.c. indicating a direct local influence of the blastocyst on the luminal epithelium. The results suggest that 1) considerable changes occur in the composition of the apical plasma membrane of the uterine epithelium when the endometrium enters the receptive state, 2) the overall trend is towards a loss of apical-type characteristics of this membrane domain and 3) the changes are modulated both systemically (by plasma steroid hormone levels) and locally by signals from the implanting blastocyst.Abbreviations d p.c. days post coitum - d. p. hCG days post hCG injection - hCG human chorionic gonadotropin - aP alkaline phosphatase - ATPase adenosine triphosphatase - Ca²⁺-ATPase Ca²⁺-activated adenosine triphosphatase - APM aminopeptidase M - GGT -glutamyl transferase - DPP IV dipeptidyl peptidase IV - PCMB p-chloromercuric benzoate - DFP di-isopropylfluorophosphate - DMF dimethylformamide     

Disturbances in the rabbit cornea after short-term and long-term wear of hydrogel contact lenses

Summary The influence of soft contact lenses (SCL) with low (37%, L) and high (65%, H) water content on rabbit corneas was investigated. The lenses were worn continuously for 1, 2, 4, 7, 10, 14, 21 or 28 days. The changes in corneal transparency, hydration and enzyme activities were studied. A slight change in corneal transparency due to higher hydration caused by a decreased activity of Na⁺–K⁺-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) in the corneal endothelium is followed by a decrease in the activity of -glutamyl transferase (GGT). Slight morphological disturbances appear within 4 days in animals wearing SCL (L). SCL (H) produce similar changes one week later. Subsequently, the corneal epithelium becomes thinner and changes in the size of corneal endothelial cells are obvious. Disturbances of enzyme activities in cells of all corneal layers are present. In the epithelium highly increased activities of acid glycosidases, acid phosphatase, and dipeptidyl peptidase I and II, in keratocytes decreased activities of alkaline phosphatase and GGT, and in the endothelium decreased activity of Na⁺–K⁺-ATPase and GGT were found. These changes are more severe after SCL (L). In this case, inflammatory cells displaying high activities of lysosomal hydrolases appear in the anterior part of the stroma during the 3rd and 4th weeks and local degradation of glycosaminoglycans and proteins takes place. In contrast, after SCL (H) a remarkable thinning of the corneas was observed during extended wear, accompanied by decreased stainability of stromal glycosaminoglycans and highly decreased enzyme activities in keratocytes. The histochemical methods proved very useful in the assessment of tesions caused by a continuous wear of SCL.     

Cytochemical localization of alkaline phosphatase in intestinal metaplasia of the human stomach

Summary Alkaline phosphatase in the brush border of areas of intestinal metaplasia of human stomach was studied cytochemically. All absorptive cells in the upper part of the villi of the duodenum had strong alkaline phosphatase activity but, in areas of intestinal metaplasia, the metaplastic glands consisted of alkaline phosphatase-positive and negative absorptive cells. Alkaline phosphatase activity was found in tall dense microvilli of absorptive cells in areas of intestinal metaplasia and in the duodenum. However, in some areas of metaplastic epithelium, the activity was very weak in some tall dense microvilli of absorptive cells but strong in those of neighbouring absorptive cells. No alkaline phosphatase activity was found in short sparse microvilli of absorptive cells in areas of intestinal metaplasia. The difference in alkaline phosphatase activity in microvilli of different cells in areas of intestinal metaplasia, which is not seen in the duodenum, indicates abnormal morphological and enzymatic differentiation in intestinal metaplasia.     

Quantitative histochemical study of alkaline phosphatase isoenzymes in the uteri of ovariectomized golden hamsters during estrogenization

The activity of two isoenzymes of alkaline phosphatase was studied in the uteri of ovariectomized golden hamsters. Animals belonging to different groups were daily injected with 10 micrograms of estrogen (octoestrol) once, for 4 and 16 days. The estrogen did not affect the overall activity lf alkaline phosphatase in the uterine luminal epithelium, but decreased the enzyme activity in stroma. Moreover, it was found that prolonged estrogen treatment increases significantly the proportion of alkaline phosphatase isoenzyme of the intestinal type in the uterine luminal epithelium.     

Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L.

The presence and localization of acid and alkaline phosphatase, non-specific proteases, aminopeptidase, amylase, non-specific esterase and lipase was investigated by histoenzymologic methods in fed and fasting turbot from day 1 to day 40 post-hatching and compared with published data. Alkaline phosphatase and aminopeptidase activities were delected at day 1 in the distal region of the developing digestive tube. At day 3 (opening of the mouth) aminopeptidase and alkaline phosphatase activities were found all along the intestine. Sites of non-specific esterase and protease activities became apparent in the digestive tract at days 2 and 3 respectively. Amylase was present in the exocrine pancreas at day 3 and in the lumen of the intestine at day 4. Acid phosphatase was active in the cellular structure surrounding the yolk stores and in the lipid droplets at day 1 and in the intestinal epithelium at day 3. Lipase was found at day 15 when the larvae metamorphose into juveniles.
All the investigated enzymes were detected in fasting animals, except for lipase. However, the intensities of the enzymatic activities were weaker in the fasting specimens relative to the fed specimens between days 7 and 10.     

Purification and partial characterization of alkaline phosphatase of matrix vesicles from fetal bovine epiphyseal cartilage. Purification by monoclonal antibody affinity chromatography

Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.     

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.     

Relocalization of membrane enzymes accompanies biliary atresia in lamprey liver

Summary Light- and electron-microscopic histochemical procedures were used to show the distribution of the membrane-bound enzymes alkaline phosphatase (Alp), adenosine triphosphatase (ATPase), and 5-nucleotidase (5-nuc) in the livers of lamprey, Petromyzon marinus, throughout the life cycle. In larvae, the three enzymes are located at the biliary pole on the canalicular membranes of microvilli. At metamorphosis the enzymes become localized at all lateral cell surfaces of hepatocytes as bile canaliculi degenerate in the programmed regression of the entire biliary tree. This latter pattern of enzyme distribution persists during the parasitic adult phase but no activity is evident in individuals in the spawning migration. As the timing of the relocalization of enzymatic activity correlates well with a build-up of bile products and iron during metamorphosis, it is suggested that the lateral surface may be the new site for transport of these products.Supported by NSERC of Canada grant no. A5945 to J.H.Y.     

Introduction of guest peptides into Escherichia coli alkaline phosphatase. Excision and purification of a dynorphin analogue from an active chimeric protein

High relative mutability may be a common property of the surfaces of all or most proteins and may be exploited during evolution not only to alter molecular recognition but to modify catalytic functions as well. Conservative amino acid substitutions often can be expected to cause minimal structural alterations, but the properties of protein surfaces and the mechanisms of protein folding that accommodate length variation without loss of function are not understood. To begin to study these aspects of protein structure and folding, we have constructed short amino acid insertions in the Escherichia coli alkaline phosphatase polypeptide by linker insertion mutagenesis of the phoA gene and have examined correlations between mutant protein function and position of the insertions relative to the x-ray map of wild type alkaline phosphatase determined by Wycoff and colleagues (Sowadski, J. M., Foster, B. A., and Wycoff, H. W. (1981) J. Mol. Biol. 150, 245-272). Mutant protein enzymatic function was generally tolerant of insertions in exterior loops, but was inactivated by insertion within alpha-helical or beta-strand structural elements. We further demonstrate that these tolerant surface loops can serve as vehicles for high level expression and stabilization of larger foreign peptide sequences, using a 15-residue analogue of dynorphin as an example. Insertion of the dynorphin "guest" peptide probably caused only a local structural perturbation of the alkaline phosphatase carrier since the hybrid protein retained enzymatic activity, was exported efficiently to the periplasmic space, and could be purified by anion-exchange chromatography using a protocol developed for alkaline phosphatase itself. The gust peptide was recovered from one of these fusion proteins intact and in high yield by protease digestion in vitro and was then purified by cation-exchange chromatography to near homogeneity in a single step.     

Electron microscope studies of phosphatases in the small intestine of Rana temporaria during larval development and metamorphosis

Summary Changes in the ultrastructure, and distribution of phosphatases in the intestinal epithelium of Rana temporaria during development were consistent with other developmental changes. Alkaline phosphatase AMP-ase and ATP-ase were always associated with sites of absorption of foodstuffs into the cell. Initially, these were only the yolk platelets but at the onset of feeding the brush border lateral wall, membranes and associated absorption vesicles all became sites of activity. At metamorphosis when the larvae cease feeding, the enzyme activities decreased and became difficult to detect.In the early larval stages, acid phosphatase activity was confined principally to the lateral cell-wall membranes. This soon disappeared but was followed at metamorphosis by a dramatic increase in both the number of sites and their activity. In general, acid phosphatase appeared to be associated with areas of degeneration. The new epithelial cells which developed during metamorphosis appeared under the old epithelium. The cell debris from the larval epithelium was then expelled into the lumen of the intestine. The new epithelium contained sites of enzyme activity similar to those of the adult. Acid phosphatase was now present only in lysosome-like bodies and very sparsely on the brush border.These results are discussed in relation to dietary and structural changes. It is suggested that the presence of the enzymes at any site can be related to and anticipate these changes, possibly under hormonal control.