Analysis of enzymatic activities of subcellular and chromatographic fractions by an automated colorimetric microassay system

Young, John Ding‐E; Damiano, Angela; Leong, Lauren Gee; Cohn, Zanvil A.

doi:10.1016/0003-2697(86)90583-x

Cited by 10 publications

(4 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Dilutions exhibiting 0.2-0.8 fractional consumption of hydrogen peroxide were used to calculate the rate (Ϫlog fractional consumption/time), and the values from different dilutions were averaged after correcting for the dilution factor. Alkaline phosphodiesterase activity was measured using a modification of a published method (21). Briefly, 3 mM p-nitrophenyl thymidine-5Ј-monophosphate sodium salt was used as substrate in 0.1 M Tris, pH 9.0, containing 2 mM zinc acetate reaction buffer, and reactions were terminated by 0.5 M glycine, pH 10.5, and absorbance was measured at 415 nm.…”

Section: Methodsmentioning

confidence: 99%

Demonstration of Lysosomal Localization for the Mammalian Ependymin-related Protein Using Classical Approaches Combined with a Novel Density Shift Method

Valle¹,

Sleat²,

Sohár³

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.Recent genomic and proteomic studies have revealed the presence of numerous previously uncharacterized proteins encoded by the mammalian genome. Determining the biological role of such proteins poses a fundamental challenge. Although sequence and structural similarities to proteins of known function may provide useful information, knowledge of the site of function is essential in understanding uncharacterized proteins. In addition to providing insight into protein function, mapping of proteins to cellular organelles or structures involved in specific biological processes may provide valuable clues to how the organelles carry out these processes.One cellular compartment that contains protein constituents of characteristic function is the lysosome, an acidic, membrane-delimited organelle present in all eukaryotic cells (1). The lysosome contains over 60 soluble proteins, most of which are hydrolases that function in concert to degrade macromolecules introduced via the endocytic and autophagic pathways into simple constituents that can be reutilized by the cell. A number of recent proteomic studies investigating soluble luminal lysosomal proteins have relied upon the fact that these proteins contain an unusual carbohydrate modification that allows for their specific affinity purification. Most luminal lysosomal proteins are t...

show abstract

Section: Methodsmentioning

confidence: 99%

Demonstration of Lysosomal Localization for the Mammalian Ependymin-related Protein Using Classical Approaches Combined with a Novel Density Shift Method

Valle¹,

Sleat²,

Sohár³

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Since the granules are lysosomal in nature, they are also enriched for a number of lysosomal markers [85]. Recent work from this laboratory has also shown that a potent neutral serine esterase activity may be used as a convenient and specific granule marker for CTL and NK cells [91,92]. The isolated granules show a morphology which is indistinct from that of intact cells ( Fig.…”

Section: Isolation Of Cytoplasmic Granulesmentioning

confidence: 98%

“…Several CTL-specific cDNA copies encoding for putative serine esterases have already been identified and cloned [41][42][43]. The serine esterase activity is contained in the granule population of these cells [91,92]. We have identified two proteins that are labeled with the specific serine esterase label [3H]DFP, migrating at 34-36 kDa and 28-30 kDa on SDS-PAGE, under reducing conditions.…”

Section: Involvement Of Other Granule Constituents In Cell Killingmentioning

confidence: 99%

“…We have identified two proteins that are labeled with the specific serine esterase label [3H]DFP, migrating at 34-36 kDa and 28-30 kDa on SDS-PAGE, under reducing conditions. Only the 34-36 kDa protein is found to have functional esterase activity, as measured using chromogenic substrates in an automated microassay system [91,92]. Under nonreducing conditions the 34-36 kDa protein migrates with a M, of 60-64 kDa.…”

Section: Involvement Of Other Granule Constituents In Cell Killingmentioning

confidence: 99%

See 1 more Smart Citation