ABSTRACT-Alkaline phosphatase (ALP) is supposed to be important for bone formation; however, its role is not clear. In this study, we examined the importance of enzymatic activity of ALP and anchoring of ALP protein to the cells for mineralization of an osteoblastic cell line, MC3T3-E1. While we cultured the cells in the presence of tetramisole, an inhibitor of ALP activity, ALP protein was expressed at a similar level to that in the control. Although tetramisole showed no effect on cell growth and increased hydroxyproline accumulation, it decreased the osteocalcin production and the accumulation of calcium and phosphate in the matrices. Tetramisole also inhibited mineralized nodule formation, which was observed by optical microscopy and detected by Von Kossa staining. On the other hand, when ALP protein was released from the cell membranes with the use of phosphatidylinositol-specific phospholipase C, no marked changes were detected in hydroxyproline, calcium and phosphate accumulations in the matrices at late calcification stage, which was consistent with the morphological findings. These results clearly show that enzymatic activity of ALP is necessary for mineralization of MC3T3-E1 cells, but not the presence of ALP protein or anchoring of ALP to the cells.Keywords: Alkaline phosphatase, Mineralization, Phosphatidylinositol-specific phospholipase C, Osteoblastic cell, TetramisoleBone-type alkaline phosphatase (ALP) is supposed to play a key role in the formation and calcification of hard tissues from accumulated circumstantial evidence (1). Although a number of theories have been proposed, its precise function is not well understood yet (2 -6). It is not even clear whether ALP is really indispensable to mineralization.ALP is attached to the external surfaces of plasma membranes by phosphoethanolamine bound to oligosaccharide, which is, in turn, covalently linked to the polar head group of phosphatidylinositol (7 -9). ALP is released from plasma membranes by phosphatidylinositol-specific phospholipase C (PIPLC) (10 -13).We previously reported that ALPs of MC3T3-E1 cells were released into the medium during cell culture and the time course of the increase of ALP in the medium was consistent with the progress of mineralization (14). We assumed that matured and released ALP might be closely related to the mineralization. An alternative report demonstrated that ALP attached covalently to the external surface of plasma membranes was involved in the mineralization of hard tissues (15).Our aim in this study was to clarify the importance of enzymatic activity of ALP and anchoring of ALP to the cells for mineralization. We used an osteoblastic cell line, MC3T3-E1 cells, which were established from fetal murine calvalial cells (16). The cells differentiate into osteoblasts and then produce ALP and process procollagens to collagens; finally the cells form mineralized tissues in vitro. We studied the effects of tetramisole, an inhibitor of ALP activity, and PIPLC additions to culture medium on mineralization of MC3T3-E1 c...
Enzyme activities such as of fructose bisphosphatase, malate dehydrogenase and carbonic anhydrase were analyzed after cytosol proteins in the mouse liver and were separated using nondenaturing two-dimensional electrophoresis (2-DE). The activities of both fructose bisphosphatase and malate dehydrogenase were inhibited by thyroxine, and fructose bisphosphatase activity was specifically inhibited by adenosine monophosphate in nondenaturing 2-DE. Furthermore, polypeptides of the separated proteins were analyzed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by peptide sequencing using electrospray ionization-tandem mass spectrometry, or both. Proteins separated by 2-DE were identified. These results indicate that the function of proteins such as enzyme activity, and their sequence structure can be analyzed, for example by peptide mapping and peptide sequencing, after the proteins have been separated by nondenaturing 2-DE. Present results also indicate analysis of enzyme activity using nondenaturing 2-DE can be applied to screen substances which affect enzyme activity.
After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.