First purification and characterization of a sucrase-isomaltase from goose kidney microvillous membrane.

Goose (Anser anser) kidney microvillus sucrase-isomaltase (EC 3.2.1.48-EC3.2.1.10) was solubilized from isolated microvillus membranes using Emulphogen BC 720 or papain. Detergent-solubilized enzyme (D-SI) was purified 149 +/- 29 times with a yield of 15.7 +/- 2.6% by a two-step procedure which included chromatofocusing. The specific activity was 2.95 +/- 0.34 U/mg protein for sucrase, 1.02 +/- 0.13 for palatinase and 5.63 +/- 0.53 for maltase. D-SI was amphiphilic as indicated by its detergent-binding properties. These properties were not observed for sucrase-isomaltase released from the microvillus membrane by papain. The Mr of the enzyme purified after solubilization by Emulphogen and papain was 543,000 and 380,000, respectively, as determined by gel filtration. The difference in Mr indicates that an Emulphogen micelle is bound to the detergent-solubilized enzyme. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis, sucrase-isomaltase migrated as several polypeptide chains: a major band (Mr 280,000) and at least seven additional minor bands (Mr 220,000-100,000). It is suggested that the major band represents the precursor pro-sucrase-isomaltase and that the lower molecular weight bands are generated by PMSF or aprotinin-resistant proteinases during homogenisation and chromatography of the enzyme. Measured by chromatofocusing, the isoelectric point was found to be pH 4.6. Sucrase-isomaltase accounts for about 20% of total microvillus membrane proteins.

Purification and characterization of kidney and intestinal brush-border membrane trehalases from the rabbit.

Trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized from the brush-border membrane of rabbit intestine and kidney by Emulphogen BC 720. The intestinal and kidney enzymes were purified 10 400-times and 4457-times respectively, in a five-step procedure, including DEAE-Trisacyl, chromatofocusing, AcA 34 gel filtration, HA Ultrogel and preparative polyacrylamide gel electrophoresis. The purified enzymes were homogeneous on polyacrylamide gel electrophoresis. The specific activities of kidney and intestinal pure trehalase were identical. Kidney and intestinal trehalases have the same molecular weight (about 85 000 by Sephadex G-200 gel filtration and 75 000 by SDS-polyacrylamide gel electrophoresis). A Stokes radius of 38 A was determined. Detergent solubilized trehalase is not retarded by phenyl-Sepharose CL-4B chromatography. The isoelectric point, measured by chromatofocusing, is between pH 3.8 and 4.2 for kidney trehalase and between pH 4.6 and 4.8 for intestinal trehalase. The enzymic properties for both kidney and intestinal trehalases are identical. The optimum pH is between 5.5 and 6.0. Trehalase is heat stable up to 50 degrees C. The apparent Km was found to be 3.5 mM in maleate buffer pH 6.0. The activation energy of trehalase is 11.17 kcal/mol. Tris, sucrose and phloridzin are fully competitive inhibitors with Ki of 3.7 mM, 3.1 mM, 1.1 mM respectively. Schiff's staining on polyacrylamide gel and interaction with Con A-Sepharose indicate that trehalase is a glycoprotein. Trehalase accounts for 0.1% and 0.3% of total brush-border membrane protein of intestine and kidney, respectively.

Studies on light-sensitive units in the deep mesencephalon of blinded frogs.

Unit responses susceptible to light stimulation of a small area on the frog's head were recorded in the deep encephalon of blinded specimens of Rana esculenta. The responses consisted of a spike discharge upon illumination. Using a threshold criterion the dark adaptation curves showed two parts, separated by a kink, the final dark threshold being complete after 20-30 min in darkness. Using a threshold criterion the spectral sensitivity curves under dark adapted conditions were broad with a peak at 548 nm. The dark adapted intensity threshold for a stimulus of 548 nm ranged between 0.15-1.4 microW/cm2.

Maltase-glucoamylase and trehalase in the rabbit small intestine and kidney brush border membranes during postnatal development, the effects of hydrocortisone.

Kidney and intestinal brush border membranes were isolated from 14-day-old rabbits and papaïn solubilized maltase-glucoamylase was purified to almost homogeneity from both membranes. Maltase-glucoamylase from kidney and intestine have the same molecular weight (669,000 daltons by AcA 22 gel filtration) and the same Km (4 mM, for maltose). Tris (Ki = 12.5 mM, for maltose) is a non-competitive inhibitor for both enzymes. In intestine, maltase and glucoamylase have low activity during the first two postnatal weeks and then undergo a sharp increase during the next 2 weeks. In contrast, for trehalase, adult levels are reached about 6 days after birth. Hydrocortisone injection to 10 days rabbits causes precocious increases in the specific activities of trehalase (3.6 x), maltase (5.2 x) and glucoamylase (7.4 x). Conversely, kidney maltase, glucoamylase and trehalase activities rise gradually from birth, reaching adult levels by the end of the third week. Administration of hydrocortisone to suckling rabbit does not affect either trehalase or maltase and glucoamylase in kidney brush border membrane.

Effect of an antiglucocorticoid (RU-38486) on hydrocortisone induction of maltase-glucosamylase, sucrase-isomaltase and trehalase in brush border membranes of suckling rats.

Induction of alpha-glycosidases by hydrocortisone in suckling rats is inhibited by the daily administration of an antiglucocorticoid (RU-38486). Conversely, RU-38486 injected daily in 15-day-old rats for 7 days does not prevent the spontaneous development of alpha-glycosidases.

Brush border membrane sucrase-isomaltase, maltase-glucoamylase and trehalase in mammals. Comparative development, effects of glucocorticoids, molecular mechanisms, and phylogenetic implications.

1. Trehalase, sucrase-isomaltase and maltase-glucoamylase are three integral glycoproteins of the brush border membranes of the enterocytes. On the basis of a comparative study on alpha-glycosidase activities (sucrase, isomaltase, maltase, glucoamylase and trehalase) associated to these glycoproteins during neonatal development, mammals could be basically divided into three groups. 2. In rodents and rabbit alpha-glycosidase activities are low or undetectable during the suckling period and increase to adult levels during the weaning period. In cat, dog and the primates examined, alpha-glycosidase activities are well or fully developed at birth. 3. In ruminants and pinnipedia alpha-glycosidases are low or absent throughout life. 4. During the suckling period of rat, mouse and rabbit, glucocorticoids trigger a premature and dramatic increase of all alpha-glycosidases. 5. On the contrary, alpha-glycosidases development during the weaning period appears to be independent of glucocorticoids. Neither hypophysectomy nor adrenalectomy prevent the development of alpha-glycosidases; only the rate of increase is reduced. 6. Transplantations of intestinal isografts either in adult or suckling animal, have shown that (1) no systemic factor inhibits the expression of alpha-glycosidase, (2) alpha-glycosidases induction is neither triggered by luminal alimentary substances, nor by hormones, (3) alpha-glycosidase development is controlled by an intrinsic ontogenic program. 7. The use of an antiglucocorticoid failed to inhibit the spontaneous development of alpha-glycosidase activities. 8. The increase of maltase and sucrase activities triggered by glucocorticoids is associated with an increase of the concentration of two glycoproteins in the microvillous membrane: sucrase-isomaltase and maltase-glucoamylase. 9. After administration of glucocorticoids the increase of maltase, sucrase and trehalase is strongly inhibited by actinomycin-D and the increase of sucrase activity is associated with a parallel increase of sucrase-isomaltase mRNA. Transcription is most likely the primary site of control of alpha-glycosidase biosynthesis. 10. In the crypt cells, alpha-glycosidases biosynthesis appears to be triggered by a receptor-mediated glucocorticoid interaction. 11. The enterocytes synthesize more alpha-glycosidase molecules as they travel to the tip of the villi. 12. The simultaneous, biosynthesis of sucrase-isomaltase and maltase-glucoamylase triggered by glucocorticoids, as well as their simultaneous normal development suggest that they may be subjected to related control mechanisms. 13. It is suggested that sucrase-isomaltase and maltase-glucoamylase might have arisen by several cycles of partial gene duplication of an ancestor gene coding for a single site maltase-isomaltase; subsequent mutation would have transformed isomaltase into sucrase or glucoamylase.

Mapping studies of the tectal representation of the frog binocular visual field. A problem of methodology.

Using the classical mapping technique, the spatio-tectal visual organization in frogs (Rana esculenta) was investigated taking special interest in the previously described existence of a "systematic disparity" between the two monocular receptive fields of rostral binocular tectal points. The existence of such a disparity was extended to the whole binocular visual field and its sign (crossed or uncrossed disparity) was studied both in paralyzed and anesthetized animals. It was proved however that this disparity phenomenon was not "systematic", but depended mainly on the experimental methodology. Its physiological significance is discussed.

Electrophysiological evidence for white light sensitivity of the encephalon in eyeless and pinealectomized frogs.

After removal of the lateral eyes and the whole pineal complex, electrophysiological responses to photic stimuli where still recorded from the diencephalon and mesencephalon of frogs. Sustained nervous discharge occurred in response to a white light stimulus while no activity was recorded in the dark.

A possible neurophysiological basis for depth perception in frogs: existence of a horopter surface.

In frogs, multi-unit receptive fields (MURF) of rostral binocular tectal points (BTP) show a crossed disparity when mapped at a distance equal to the perimeter radius (i.e., 33 cm). The shape of the spatial surface where MURF of all BTP are in-register is investigated in two planes: (a) in the longitudinal plane, the locus of superimposition is a circumference passing through both eyes; (b) in the vertical plane, it corresponds to a straight line tilted towards the animal's head. This surface can be defined as the frog's horopter surface since it represents the spatial locus where objects can simultaneously stimulate corresponding retinal areas. Behavioural and electrophysiological correlations are discussed.

Rat intestinal brush border membrane trehalase: some properties of the purified enzyme.

Rat intestinal brush border trehalase (EC 3.2.1.28) solubilized by Triton X-100 or Emulphogen BC 720 has been purified almost to homogeneity in a five steps procedure including DEAE cellulose, Sephadex G-200, preparative flat bed electrofocusing and hydroxylapatite. The apparent molecular weight was estimated to be about 65,500 daltons by mannitol density gradient ultracentrifugation. The optimum pH of the enzyme was between 5.5 and 5.7 in phosphate, maleate or citrate buffers. The apparent Km for trehalose was found to be 10 mM in maleate buffer pH 6.0. The isoelectric point was 4.9. Tris, P-aminophenylglucoside, sucrose and maltose are fully competitive inhibitors with Kis of 2.2, 1.8, 7.7 and 170 mM, respectively. The inhibition by Phloridzin appeared to be of the mixed type with a Ki of 1.7 mM. Trehalase is heat stable up to 50 degrees C and the activation energy is 10.96 kcal/mol. Schiff's staining on polyacrylamide gel and interaction with Con-A-Sepharose indicate that rat trehalase is a glycoprotein.

[Topography of the tecto-tectal component of the main ipsilateral visual pathway of the frog (Rana esculenta L.)]. / Topographie de la composante tecto-tectale de la voie visuelle ipsilatérale principale de la Grenouille (Rana esculenta L.).

The detailed topography of the tecto-tectal component of the Frog's ipsilateral visual pathway is electrophysiologically obtained by mapping the optic lobes. This linkage transfers the visual information explored along a transversal tectal row on to an homologous line oriented at 130 degrees on the opposite tectum. The ipsilateral projection of the temporo-nasal axis of the retina, but not of the antero-posterior axis of the visual field, is reversed compared to its contralateral projection. Finally, the majority of the homologous tectal points are asymmetrical with respect to the animal's sagittal axis.

Diencephalic binocular wide field neurons in the frog.

Single binocular neurons were recorded in the frog diencephalon (area dorsalis posterior and area ventralis thalami). These neurons respond to any moving stimulus from 1 degree 30 to 32 degrees in diameter, without directional selectivity and to an ON-OFF light stimulation. They are not activated by stationary objects. Habituation is also commonly observed. The most important feature of these neurons is their wide receptive field which covers the whole visual field of the frog. Evidence that these neurons receive inputs from each tectum is discussed.

[Existence of a non-retinotopic contralateral retino-tectal visual projection in the normal frog Rana esculenta L]. / Existence d'une projection visuelle rétino-tectale contralatérale non rétinotopique chez la grenouille normale. Rana esculenta L.

In the Frog, after unilateral optic nerve and tract section, contralateral visual responses were recorded in the tectum ipsilateral to the section. These responses were elicited by stimulation of a unique spatial region located near the projection of the contralateral eye optic axis and could be randomly recorded on the tectal surface. The possible pathways and the role of such retino-tectal afferences are discussed.

The influence of hydrocortisone on the synthesis and turnover of microvillous membrane glycoproteins in suckling rat intestine.

Synthesis and degradation of intestinal mucosal and microvillous membrane glycoproteins were studied in control suckling rats, and suckling rats given cortisol acetate by intraperitoneal injection for 3 days. Cortisol acetate had no effect on total uptake of radioactive glucosamine by the protein free compartment of rat intestine. Early incorporation of [1(-14)C]glucosamine by intestinal glycoproteins was enhanced by cortisol, but stimulation was the same in membrane and homogenate fractions. Polyacrylamide gel electrophoresis of membrane proteins solubilized with 2% sodium dodecyl sulphate demonstrated a cortisol dependent change, characterized by loss of faster travelling glycoproteins, and a corresponding shift in maximum labelling at 3 h from these glycoproteins to more slowly migrating glycoproteins. Degradation was studied qualitatively with a double isotope technique. Glycoprotein degradative rates appeared to be stimulated by cortisol, but similarly in membrane and total homogenate fractions. On polyacrylamide gels, the areas occupied by glycoproteins with the highest apparent degradative rates, corresponded closely with the areas of most active labelling at 3 h. The rate of degradation in the most actively labelled zone appeared to be higher after cortisol than in the controls. The results indicate that cortisol does not alter membrane composition by inhibiting degradation of selected glycoproteins, and are consistent with a model in which cortisol stimulates the synthesis of specific membrane glycoproteins in suckling rats, while inhibiting synthesis of other glycoproteins.

Pineal response types in the frog's brain under white light exposure.

Responses to white light stimulation can be recorded with electrophysiological methods at the level of (1) the pineal system (2) different diencephalic nuclei and (3) the mesencephalic tegmentum. The neurons are classified in six groups according to their discharge characteristics. As some responses present a higher complexity than the classical messages, an integration of the pineal informations during their course toward the brain is suggested in order to support phototactic behavior.

Isolation of microvillus plasma membranes from suckling-rat intestine. The influence of premature induction of digestive enzymes by injection of cortisol acetate.

1. Cortisone administration to suckling rats leads prematurely to induction of enzymes of the intestinal microvillus plasma membrane and lengthening of the intestinal microvilli. To investigate the membrane changes that might be involved, a method for the isolation of a fraction enriched with microvillus plasma membrane was developed in suckling rats. Plasma-membrane fractions were compared from 13-day-old control rats and from 13-day-old rats given cortisol acetate by subcutaneous injection for 3 days. 2. After cortisol injection, the activity of maltase, trehalase, sucrase and leucyl beta-naphthylamidase increased markedly, and to the same extent, in intestinal homogenates and plasma-membrane preparations. Purification, and recovery of five marker enzymes with respect to homogenate activity, and recovery of protein, were similar for both membrane preparations, particularly after correction for non-membrane activity, which was high in suckling rats and affected by cortisol. 3. In material released from the plasma membrane by digestion with papain, maltase protein was increased after cortisol injection at least as much as maltase activity. Sucrase activity increased at least 200-fold, and this increase was associated with the appearance of a new sucrase band on polyacrylamide-gel electrophoresis. 4. Sodium dodecyl sulphate electrophoresis of plasma-membrane proteins revealed at least four additional macromolecules after cortisol injection. Concurrently several proteins disappeared from the plasma membrane. The added proteins appeared in the main to be removed from the plasma membrane by papain, whereas the deleted proteins were in the papain-resistant fraction. 5. Enzymic stimulation induced by cortisol acetate in the suckling-rat plasma membrane therefore appears to involve the addition of new proteins, rather than activation of proteins in situ. Deletion of proteins from the membrane during induction of hydrolytic enzymes may reflect other phenomena such as protein reorganization associated with the change in microvillus shape.