Specific Assays of some Phosphatases in Subcellular Fractions of Small Intestinal Mucosa

Hübscher, G.; West, Gwen R.

doi:10.1038/205799a0

Cited by 432 publications

(76 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3). The patterns of 14C-fatty acids were quite similar in all the fractions: palmitate, 17 to 27 %; stearate, 49 to 62%; and very long chain saturated (C20-C28), 21 Since the rate of germination varied widely between different species, a morphological criterion for comparison was adopted. Thus the length of exposure to acetate-'4C was determined by the time required for the dry seed to imbibe water and develop to the emergence of the radicle through the seed coat.…”

Section: Resultsmentioning

confidence: 99%

Fat Metabolism in Higher Plants

Harwood¹,

Stumpf²

1970

Plant Physiol.

View full text Add to dashboard Cite

To understand more fully organelle membrane assemblage, the synthesis of the first fatty acids by the germinating pea, Pisum sativum, was studied by the incorporation of either tritiated water or acetate-1_'4C into lipids by the intact, initially dry seed. After a lag phase, labeling proceeded linearly. This lag phase ended when uptake of water had increased the seed weight to 185% of its original weight. The first fatty acids synthesized were palmitic and stearic followed shortly after by long chain saturated fatty acids (C20-C26). The synthesis of very long chain acids was consistently characteristic of several other seeds in early stages of germination. The majority of the radioactive acids were present in phospholipids and were localized in particulate fractions. The acyl components of phosphatidyl glycerol were highly labeled. (13) showed a relatively unspecific breakdown of stored fat.The use of tritiated water as a source of label for newly synthesized fatty acids provides a good alternative to the commonly used acetate. Spedding and Wilson (39) have used this isotope to study amino acid metabolism at very early stages of germination of mustard, and Foster and Katz (9) studied its incorporation into fatty acids by rat adipose tissue. Since water is intimately involved with the natural germination process, use of tritium oxide does not suffer from the criticism of using an artificial substrate. The results of its use can then be compared to the use of acetate, in case of differences.Since pea seeds are readily germinated with concomitant lipid synthesis (32), they were chosen to study the synthesis of fatty acids at very early times of germination. It is at this stage that reorganization of cell components occurs, directed to organelle formation. Thus this paper attempts to define the nature of the fatty acids and complex lipids which are synthesized by the seed as germination begins.Germination of a plant seed involves a complex series of metabolic processes such as water imbibition, respiration, nucleic acid and protein synthesis, mobilization of food reserves as well as cell differentiation and growth (33). Plant fatty acid synthesis has been studied with peas by Macey and Stumpf (32). These low lipid, high starch-containing seeds were found to incorporate acetate-1-14C into fatty acids from C16 to C28. The enzyme systems responsible for these syntheses were further studied and evidence was presented that the microsomal fraction participated in the synthesis of the very long chain fatty acids, probably from malonyl-acyl carrier protein. Either green or etiolated barley leaves also synthesized fatty acids of chain length up to C24 (17). The same authors (18) found that these acids were located in a particulate cellular fraction. Kolattukudy (28), using Brassica leaf tissue, found very long chain fatty acids produced from acetate.Partially purified enzyme preparations of fatty acid synthetase usually produced a pattern of fatty acids different from the pattern found with the endogenous fatty acid...

show abstract

Section: Resultsmentioning

confidence: 99%

Fat Metabolism in Higher Plants

Harwood¹,

Stumpf²

1970

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…ß-Glucuronidase was assayed as described by Fishman and Bernfeld (16) . Glucose-6-Pase was determined by the method of Hubscher and West (17), except that the reaction contained 0.01 M glucose-6-P and 0.1 M maleic acid buffered at pH 6.5 in a total volume of 0.4 ml . The reaction was incubated at 37°C for 10 min and stopped with 0 .1 ml of 12% HC1O4.…”

Section: Met110dsmentioning

confidence: 99%

Isolation and Biochemical Characterization of Brush Borders From Rabbit Kidney

Berger

Sacktor

1970

The Journal of Cell Biology

152

View full text Add to dashboard Cite

A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated . The procedure was monitored by phase and electron microscopy and marker enzymes, i .e . ATP :NMN adenylyl transferase, nuclear ; cytochrome oxidase, mitochondrial ; (3-glucuronidase, lysosomal ; and glucose-6-Pase, microsomal ; and indicated an essentially pure preparation of brush borders . The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders . Maltase was also found ; the specific activities of the two enzymes in the brush borders were increased 10-to 20-fold . Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent . It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na+ + K+) ATPase, an oligomycin-insensitive Mg++ ATPase, and a Ca++-activated ATPase . Alkaline phosphatases, dephosphorylating (3-glycero-P, and trehalose-6-P were also present . The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations ; however, activities were found in other subcellular fractions of the renal cortex . Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions . Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex .The renal brush border has a prominent role in determining the specificity and rate of reabsorption from the glomerular filtrate . A characterization of the biochemical properties of the isolated brush border, therefore, should provide significant information on the molecular mechanisms underlying tubular reabsorption . Brush borders from intestinal epithelial cells have been isolated as a discrete subcellular fraction, and several enzymes are now known to be associated with this membranous structure (1-4) . In contrast, little has been done on the isolation or biochemical characterization of the renal brush border . Recently, however, Thuneberg and Rostgaard (5) described a procedure for isolating brush borders from rabbit kidney cortex, but no biochemical correlates were made, and Kinne and Kinne-Saffran (6) and

show abstract

“…For fi-galactosidase (EC 3.2.1.23) the method of Sellinger et al (1960) was used. Glucose 6-phosphatase (EC 3.1.3.9) was assayed in the presence of 4mM-EDTA and 2mM-KF as described by Hubscher & West (1965). Stetten & Ghosh (1971) found that the liver enzyme was stimulated by preincubation with NH4+ ions.…”

Section: Methodsmentioning

confidence: 99%

“…Hubscher & West (1965) have shown that EDTA inhibits alkaline phosphatase and NaF inhibits acid phosphatase in cat intestine. To show whether these inhibitors are also specific for the rat kidney enzymes, alkaline and acid phosphatase were assayed in their presence with 2-glycerophosphate as substrate.…”

Section: Elimination Ofacid and Alkaline Phosphatase Activitiesmentioning

confidence: 99%

Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

Cooper

Hawthorne

1975

Biochemical Journal

View full text Add to dashboard Cite

The properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergents sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the substrate and in the presence of Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and Li+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and densitygradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that ofthe liver Golgi marker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble. Sloane-Stanley (1953) showed that brain phosphoinositide fractions were rapidly attacked by brain homogenates with the release of free and combined phosphate as well as free and combined inositol. Rodnight (1956) later performed experiments to suggest that two enzymes, a phosphomonoesterase and a phosphodiesterase, were present. Work with pure triphosphoinositide (phosphatidyl-myoinositol 4,5-bisphosphate)$ indicated that the lipid was broken down first to diphosphoinositide (phosphatidyl-myo-inositol 4-phosphate) and then to phosphatidylinositol

show abstract

Specific Assays of some Phosphatases in Subcellular Fractions of Small Intestinal Mucosa

Cited by 432 publications

References 4 publications

Fat Metabolism in Higher Plants

Fat Metabolism in Higher Plants

Isolation and Biochemical Characterization of Brush Borders From Rabbit Kidney

Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

Contact Info

Product

Resources

About