A quantitative colorimetric method of measuring alkaline phosphatase activity in eukaryotic cell membranes

Akçakaya, Handan; Aroymak, Aysin; Gökçe, Sina

doi:10.1016/j.cellbi.2006.11.014

Cited by 31 publications

(17 citation statements)

References 31 publications

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“…ALP activity of MC3T3-E1 osteoblasts was determined by a modified colorimetric enzyme assay [19]. After culture protocols, cells were lysed with 0.2% Triton X-100, and the lysates were centrifuged at 14,000 ×g for 10 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Antiosteoclastic Activity of Milk Thistle Extract after Ovariectomy to Suppress Estrogen Deficiency-Induced Osteoporosis

Kim

Kang

et al. 2013

BioMed Research International

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Bone integrity abnormality and imbalance between bone formation by osteoblasts and bone resorption by osteoclasts are known to result in metabolic bone diseases such as osteoporosis. Silymarin-rich milk thistle extract (MTE) and its component silibinin enhanced alkaline phosphatase activity of osteoblasts but reduced tartrate-resistant acid phosphatase (TRAP) activity of osteoclasts. The osteoprotective effects of MTE were comparable to those of estrogenic isoflavone. Low-dose combination of MTE and isoflavone had a pharmacological synergy that may be useful for osteogenic activity. This study attempted to reveal the suppressive effects of MTE on bone loss. C57BL/6 female mice were ovariectomized (OVX) as a model for postmenopausal osteopenia and orally administered 10 mg/kg MTE or silibinin for 8 weeks. The sham-operated mice served as estrogen controls. The treatment of ovariectomized mice with nontoxic MTE and silibinin improved femoral bone mineral density and serum receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio, an index of osteoclastogenic stimulus. In addition, the administration of MTE or silibinin inhibited femoral bone loss induced by ovariectomy and suppressed femoral TRAP activity and cathepsin K induction responsible for osteoclastogenesis and bone resorption. Collectively, oral dosage of MTE containing silibinin in the preclinical setting is effective in preventing estrogen deficiency-induced bone loss.

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Section: Methodsmentioning

confidence: 99%

Antiosteoclastic Activity of Milk Thistle Extract after Ovariectomy to Suppress Estrogen Deficiency-Induced Osteoporosis

Kim

Kang

et al. 2013

BioMed Research International

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“…The following marker enzyme assays were used to detect specific subcellular compartments: Alkaline phosphatase for the plasma membrane [20], Succinate dehydrogenase for mitochondria [21] and Cytochrome c reductase (NADPH) for the endoplasmic reticulum (Cytochrome c Reductase (NADPH) Assay Kit, Sigma-Aldrich, St.Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

2010

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BackgroundThe plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila.ResultsWe show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.ConclusionA combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.

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“…MEASUREMENT OF ALP ACTIVITY ALP activity of osteoblastic MC3T3-E1 cells was measured by a modified colorimetric enzyme assay [Akcakaya et al, 2007]. After culture protocols, cells were lysed with 0.2% Triton X-100 and the lysates were centrifuged at 14,000g for 10 min at 48C.…”

Section: Measurement Of Ecm Calcium Depositsmentioning

confidence: 99%

Osteoblastogenesis and osteoprotection enhanced by flavonolignan silibinin in osteoblasts and osteoclasts

Kim

Kang

et al. 2011

J of Cellular Biochemistry

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Bone-remodeling imbalance induced by decreased osteoblastogenesis and increased bone resorption is known to cause skeletal diseases such as osteoporosis. Silibinin is the major active constituent of silymarin, the mixture of flavonolignans extracted from blessed milk thistle (Silybum marianum). Numerous studies suggest that silibinin is a powerful antioxidant and has anti-hepatotoxic properties and anti-cancer effects against carcinoma cells. This study investigated that silibinin had bone-forming and osteoprotective effects in in vitro cell systems of murine osteoblastic MC3T3-E1 cells and RAW 264.7 murine macrophages. MC3T3-E1 cells were incubated in osteogenic media in the presence of 1-20 µM silibinin up to 15 days. Silibinin accelerated cell proliferation and promoted matrix mineralization by enhancing bone nodule formation by calcium deposits. In addition, silibinin furthered the induction of osteoblastogenic biomarkers of alkaline phosphatase, collagen type 1, connective tissue growth factor, and bone morphogenetic protein-2. Differentiated MC3T3-E1 cells enhanced secretion of receptor activator of nuclear factor-κB ligand (RANKL) essential for osteoclastogenesis, which was reversed by silibinin. On the other hand, RAW 264.7 cells were pre-incubated with 1-20 µM silibinin for 5 days in the presence of RANKL. Non-toxic silibinin markedly attenuated RANK transcription and intracellular adhesion molecule-1 expression elevated by RANKL, thereby suppressing the differentiation of macrophages to multi-nucleated osteoclasts. It was also found that silibinin retarded tartrate-resistant acid phosphatase and cathepsin K induction and matrix metalloproteinase-9 activity elevated by RANKL through disturbing TRAF6-c-Src signaling pathways. These results demonstrate that silibinin was a potential therapeutic agent promoting bone-forming osteoblastogenesis and encumbering osteoclastic bone resorption.

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A quantitative colorimetric method of measuring alkaline phosphatase activity in eukaryotic cell membranes

Cited by 31 publications

References 31 publications

Antiosteoclastic Activity of Milk Thistle Extract after Ovariectomy to Suppress Estrogen Deficiency-Induced Osteoporosis

Antiosteoclastic Activity of Milk Thistle Extract after Ovariectomy to Suppress Estrogen Deficiency-Induced Osteoporosis

Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

Osteoblastogenesis and osteoprotection enhanced by flavonolignan silibinin in osteoblasts and osteoclasts

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