A Method for the Ultrastructural Localization of Sucrase

1972 ◽  
Vol 30 ◽  
pp. 6-7
Author(s):  
V. Lorenzsonn ◽  
H. Agresti ◽  
W. A. Olsen
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Ultrastructural Localization ◽  
Negative Staining ◽  
Sucrase Activity ◽  
Alternative Method ◽  
Brush Borders ◽  
Surface Localization ◽  
Microvillus Membrane ◽  
Intestinal Sucrase

Several studies have suggested that intestinal sucrase is located in 60 Å particles of microvillus membrane seen with negative staining. This localization has been questioned, however, because of a disparity between the disappearance of particles and enzyme activity from brush borders during 20 min. papain digestions. Ferritin labeled antibody to sucrase has also been used to study surface localization of the enzyme. We have devised an alternative method to allow us to study intracellular localization as well for study of the increased sucrase activity in diabetic mucosa. We modified the technique of Keston for ultrastructural use by replacing the chromagen with 3,3'-diaminobenzidine tetrahydrochloride (DAB).

Precocious and reversible expression of sucrase-isomaltase unrelated to intestinal cell turnover

1994 ◽  
Vol 266 (4) ◽  
pp. G568-G575 ◽  
Author(s):  
E. Nsi-Emvo ◽  
C. Foltzer-Jourdainne ◽  
F. Raul ◽  
F. Gosse ◽  
I. Duluc ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Epithelial Cell ◽  
Serum Level ◽  
Intestinal Cell ◽  
Epithelial Cell Proliferation ◽  
Cell Turnover ◽  
Sucrase Activity ◽  
Intestinal Sucrase

The effect of starvation and refeeding on the developmental pattern of intestinal sucrase-isomaltase (SI) was analyzed in preweaned rats. Starvation at postnatal day 12 caused a precocious expression of SI activity and mRNA. Alkaline phosphatase activity was slightly reduced, and no significant change was observed for aminopeptidase and lactase activities. Immunostaining showed that SI molecules appear in cells at the base of the villus. Sucrase expression was further increased by prolonged food deprivation, whereas enzyme activity as well as the amount of SI mRNA dropped to reach the low level found in control sucklings when 48 h-starved pups were refed by returning them to their dams. During the refeeding period, the enterocytes that were committed to produce SI by starvation continued to express the enzyme while migrating up the villi. However, the new epithelial cells arising from the crypts no longer synthesized the disaccharidase. The starvation-evoked appearance of SI was preceded by a transient burst of expression of the protooncogene c-fos, an event that may be correlated to the ontogenic rise of c-fos mRNA observed before weaning. However, in contrast to the normal weaning condition, SI induction by starvation occurred without obvious increase of epithelial cell proliferation and turnover. During the starvation and refeeding period, patterns of sucrase activity and SI mRNA paralleled the serum level of glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF Mg++-DEPENDENT DINITROPHENOL-STIMULATED ADENOSINE TRIPHOSPHATASE IN HUMAN BLOOD PLATELETS

1967 ◽  
Vol 15 (5) ◽  
pp. 267-272 ◽  
Author(s):  
VICTOR G. VETHAMANY ◽  
SYDNEY S. LAZARUS
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Human Blood ◽  
Adenosine Triphosphatase ◽  
Blood Platelets ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Structural Localization ◽  
Adenosine Triphosphatase Activity ◽  
Human Blood Platelets

Fine structural localization of adenosine triphosphatase activity was studied in human platelets briefly fixed in cold formol calcium and then incubated in lead medium with added dinitrophenol. Under these conditions, the Mg++-dependent dinitrophenol-stimulated adenosine triphosphatase of platelet mitochondria was demonstrated, but neither granules nor plasma membrane showed enzyme activity.

Ultrastructural localization of cationic proteins in cytoplasmic granules of chicken and rabbit polymorphonuclear leukocytes

1975 ◽  
Vol 17 (1) ◽  
pp. 79-94
Author(s):  
E.K. Macrae ◽  
J.K. Spitznagel
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Intracellular Localization ◽  
Ultrastructural Localization ◽  
Cationic Protein ◽  
Cationic Proteins ◽  
Cytoplasmic Granules ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Neutrophilic Polymorphonuclear Leukocytes ◽  
Ammoniacal Silver

Cytoplasmic granules known to contain cationic arginine-rich proteins can be identified by the ammoniacal silver reaction (ASR) which provides a cytochemical marker detectable under the electron microscope. Only the large rod-shaped granules of the chicken polymorphonuclear leukocytes (heterophils) and the large spherical azurophilic granules of the rabbit neutrophilic polymorphonuclear leukocytes show the ASR product as a discrete particulate electron-dense deposit. The other smaller granules are devoid of reaction product, as are membranes and mitochondria. The intracellular localization of the ASR product, as are membranes and mitochondria. The intracellular localization of the ASR product on the large granules coincides with the ASR product localization on the same isolated granule populations, when the ammoniacal silver reaction is applied to these granules after their separation by sucrose-density gradients. The cationic proteins may have intraleukocytic bacteriolytic properties, since ASR product, presumably indicating cationic protein from discharged granules, appears to surround ingested bacteria within cytoplasmic phagosomes.

Development and tissue distribution of sucrase-isomaltase mRNA in rats

1990 ◽  
Vol 258 (1) ◽  
pp. G52-G58 ◽  
Author(s):  
L. L. Leeper ◽  
S. J. Henning
Keyword(s):  
Enzyme Activity ◽  
Small Intestine ◽  
Northern Blot ◽  
Blot Analysis ◽  
Molecular Basis ◽  
Northern Blot Analysis ◽  
Sucrase Activity ◽  
Old Rats ◽  
Two Parameters ◽  
Precocious Development

Previous studies of sucrase-isomaltase (SI) activities have shown this complex to be absent in the suckling rat and to appear during the weaning period. We describe here the cloning of a heterologous SI cDNA and its use for the quantitation of SI mRNA as a first step toward understanding the molecular basis of SI development. A survey of RNA from 12 tissues of mature rats by Northern blot analysis showed a 6-kb band of SI mRNA only in the small intestine. Within the latter, both sucrase activity and SI mRNA peaked in the jejunum. Assay of jejunal tissue from developing rats showed sucrase activity and SI mRNA to be first detectable at 18 days, to rise in parallel through 24 days, and then to diverge a little (enzyme activity being lower) by 36 days. When glucocorticoid was administered to 10-day-old rats, neither sucrase activity nor SI mRNA was detectable 12 h later. Both parameters were readily detected 24 h postinjection, although the mRNA had risen relatively more than the enzyme activity. The two parameters increased in concert through 5 days postinjection and then plateaued. We conclude that, with respect to distribution along the intestine and to normal and precocious development, activities of SI in the rat are determined primarily by the abundance of its mRNA.

RAT INTESTINAL SUCRASE: II. THE EFFECTS OF RAT AGE AND SEX AND OF DIET ON SUCRASE ACTIVITY

1963 ◽  
Vol 41 (1) ◽  
pp. 917-929 ◽  
Author(s):  
D. G. R. Blair ◽  
W. Yakimets ◽  
J. Tuba
Keyword(s):  
Control Diet ◽  
Male Rats ◽  
Normal Male ◽  
Casein Diet ◽  
Sucrase Activity ◽  
Adult Rats ◽  
Sucrose Diet ◽  
Enzyme Adaptation ◽  
Intestinal Sucrase ◽  
Intestinal Sucrase Activity

Intestinal sucrase activity of the rat varies with the age, but not the sex, of the animal. Sucrase activity of rats 23 days of age was approximately two-thirds that of adults.Sucrase activity of adult rats was significantly decreased by several days of fasting. The decrease was rapid during the first 2 to 4 days of the fast, but became negligible thereafter.Diets containing large (70%) amounts of sucrose, galactose, melizitose, or α-methyl-D-glucoside produced highly significant increases in intestinal sucrase levels (compared with a carbohydrate-free, high-casein control diet) when fed ad libitum for 24 hours to adult male rats previously fasted for 3 days. Similar diets containing fructose, fructose plus glucose in equimolar amounts, or maltose significantly increased sucrase activity, but diets containing glucose, mannose, xylose, or lactose were not stimulatory. A 70% raffinose diet significantly decreased sucrase activity. Normal male rats which were fed the 70% sucrose diet for 4 weeks had sucrase activities similar to those of controls fed Purina fox checkers, but animals fed the carbohydrate-free, high-casein diet for 1 day or longer had sucrase activities significantly lower than those of controls. The significance of these observations in regard to enzyme "adaptation" is discussed.

scholarly journals The effects of l-arabinose on intestinal sucrase activity: dose-response studies in vitro and in humans

2011 ◽  
Vol 94 (2) ◽  
pp. 472-478 ◽  
Author(s):  
Inger Krog-Mikkelsen ◽  
Ole Hels ◽  
Inge Tetens ◽  
Jens Juul Holst ◽  
Jens Rikardt Andersen ◽  
...  
Keyword(s):  
Dose Response ◽  
Sucrase Activity ◽  
Intestinal Sucrase ◽  
Intestinal Sucrase Activity

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

scholarly journals Qualitative difference of subcellular localization of tumor-associated carbohydrate (Le(x)) antigens in renal cell carcinoma and normal kidney.

1991 ◽  
Vol 39 (4) ◽  
pp. 479-484 ◽  
Author(s):  
H Ohtani ◽  
Y Fukushi ◽  
S Orikasa ◽  
H Nagura
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Plasma Membranes ◽  
Clear Cell ◽  
Clear Cell Carcinoma ◽  
Intracellular Localization ◽  
Qualitative Difference ◽  
Ultrastructural Localization ◽  
Normal Kidney

Renal cell carcinomas are immunohistochemically positive for oligosaccharides with the Le(x) determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and its derivatives, as oncofetal antigens, and their expression is closely related to a better prognosis of the patients. This study was designed to clarify the difference in antigen localization at the ultrastructural level between renal cell carcinoma and normal tissues. In normal kidneys, Le(x) detected by monoclonal antibody (MAb) FH 2 and sialylated extended Le(x) (sialyl Le(x)-i) by MAb FH 6 were identified along the plasma membrane of microvilli of proximal tubule epithelial cells, with occasional immunoreactivity along the basolateral plasma membranes. Intracellular localization was very sparse. Renal cell carcinoma showed localization of Le(x) and sialyl Le(x)-i antigens along the cell membrane and in the cytosol as aggregates or filaments. Immunoreactive materials were also observed in the lumen formed among carcinoma cells. The cytosolic immunoreactivity, not observed in the normal kidney, was regarded as "abnormal cytosolic accumulation" of the antigens. This pattern was more pronounced in clear-cell carcinoma. Pretreatment of specimens with chloroform-methanol, which extracts glycolipids, decreased immunoreactivity in carcinoma tissues, particularly that in the cytosol. The extracts contained substances immunoreactive for MAb FH6. Our study has demonstrated that (a) remarkable changes occur in the ultrastructural localization patterns of sialyl Le(x)-i and Le(x) in renal cell carcinoma and (b) considerable amounts of glycolipids are contained in the substances with sialyl Le(x)-i deposited in the cytosol of clear-cell carcinoma.

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