Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We addressed the question to what extent alkaline phosphatase (ALP) can induce mineralization of a collagenous matrix implanted subperiosteally, and how the graft interacts with the underlying bone. Bovine intestinal ALP was bound to sheets of guanidine-extracted, demineralized bovine dentin by using the crosslinking agent 1-ethyl-3(3-dimethylaminopropyl)carbodiimide.HCl. The complexes (with active enzyme) and control grafts (no enzyme) were implanted over osseous defects in opposite halves of rat calvaria. After time intervals varying from 3-12 weeks, the calvaria were processed for light and electron microscopic examination and histomorphometric analysis. The ALP-containing sheets (but not their controls) rapidly accumulated mineral crystals. As the complexes mineralized, osteoblasts appeared and formed a layer of bone in direct contact with the grafted material. The results indicate that ALP induced the deposition of mineral crystals, and strongly suggest that it is this mineral component which influenced the formation of bone.
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PMID:Mineralization of dentinal collagen sheets complexed with alkaline phosphatase and integration with newly formed bone following subperiosteal implantation over osseous defects in rat calvaria. 845 21

It is well known that the bone matrix contains proteins which can induce ectopic endochondral bone formation in vivo. One class of these proteins is the bone morphogenetic protein (BMP). In order to investigate the physiological function of the BMP, its purification was attempted from an extract of demineralized bone matrix and its actions on the osteoblastic cell line were investigated. To isolate the BMP, a demineralized bone matrix was extracted with 4M guanidine-HCl. A water-insoluble fraction (G-WI) was separated from the demineralized bone extract by dialysis against distilled water and centrifugation. The BMP was purified from G-WI by gel filtration on Sephacryl S-200 HR, cation exchange with Mono-S, heparin affinity column and finally by C1/8 reverse phase chromatography. Peptide sequence analysis revealed that the purified BMP fraction contained "BMP-3" reported by Wozney et al. (1988). In order to investigate its function, the BMP was applied to the rat osteogenic sarcoma cell line UMR108. The BMP inhibited the growth of the UMR108 cells and enhanced the alkaline phosphatase activity in a dose-responsive manner.
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PMID:[Purification of bone morphogenetic protein and investigation of its effects on osteoblastic cell line UMR108]. 848 1

Acid-induced and guanidine hydrochloride (GdnCl)-induced reversible unfolding of Escherichia coli alkaline phosphatase (AP) was characterized under equilibrium conditions. The protein was exposed to extreme conditions of pH 2.0 or 6 M GdnCl and was subsequently returned to normal conditions. Associated changes in the protein structure was probed by various spectroscopic methods. The changes in the functional properties were monitored by measuring enzymatic activity, capacity to renature spontaneously upon removal of the denaturant, and renaturation in presence of various site-specific and nonspecific effector molecules, in the absence and presence of beta-mercaptoethanol. Analysis of the fluorescence and CD spectra showed that the unfolding of the organized structures was much more extensive in 6 M GdnCl than at pH 2.0. Intrachain S-S bonds in each unfolded state were accessible to reduction by beta-mercaptoethanol. The effectors Zn2+ and ATP induced renaturation of active site only under reducing conditions, whereas Triton X-100 or alpha-crystallin needed the presence of some organized structure. The reconstituted protein from each denatured state without or with an effector showed different CD spectra. It is concluded that the active site domain of AP could be reconstituted independently of other structural domains in different pathways.
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PMID:Reversible unfolding of Escherichia coli alkaline phosphatase: active site can be reconstituted by a number of pathways. 865 92

When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.
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PMID:In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state. 889 9

A binding enzyme-linked immunosorbent assay (ELISA) has been developed for measuring nanogram concentrations of semisynthetic pneumocandin antifungal agents in human plasma. Semisynthetic pneumocandin L-733,560 was conjugated to succinylated hemocyanin by water-soluble carbodiimide and was used as an immunogen to produce polyclonal antibodies in rabbits. Pneumocandins were used to directly coat the wells of a microtiter plate, and quantitation was achieved by using rabbit polyclonal antibodies to pneumocandin L-733,560 and goat anti-rabbit immunoglobulin G conjugated to either alkaline phosphatase or horseradish peroxidase. Maximum binding of L-733,560 and most related analogs to the wells of the microtiter plate was found to occur in the first 5 min of incubation at 4 degrees C. Once bound to the plate, these pneumocandins could not be removed from the plate, either by treatment with 4.0 to 6.0 M urea or by treatment with 4.0 to 6.0 M guanidine hydrochloride for 24 h at 4 degrees C. The binding ELISA is linear with drug concentration and can detect levels of L-733,560 as low as 5 ng/ml in human plasma. The assay is also useful for quantitating plasma levels of related semisynthetic pneumocandins including clinical candidate MK-0991.
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PMID:Novel enzyme-linked immunoassay to determine nanogram levels of pneumocandins in human plasma. 957 17

The in vitro folding of Escherichia coli alkaline phosphatase (AP) from the guanidine hydrochloride (GdnHCl) denatured state is characterized by a significant slow phase in the post activational recovery of native protein lability (probed by the susceptibility to GdnHCl denaturation and occurring on the time scale of days) as well as a slow phase in the recovery of activity (on the time scale of minutes). Slow folding events have often been attributed to cis-trans isomerizations of X-Pro peptide bonds, a plausible explanation for AP, which contains 21 prolines per subunit. To investigate the role of proline isomerization in the two measures of refolding mentioned above, we have performed "double-jump" GdnHCl denaturation/renaturation experiments, with a third jump, where the rate of unfolding of refolded protein upon exposure to denaturant was added to assess the rate of change of lability. Our measurements of the time evolution of both the lability and the reactivation of refolded AP as a function of denaturation time show that proline isomerization is unlikely to be the cause of either of these slow events in the refolding of AP. The conclusions are further confirmed by the absence of proline isomerization effects when AP is refolded in the presence of human and periplasmic E. coli peptidyl-prolyl isomerase.
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PMID:Proline isomerization is unlikely to be the cause of slow annealing and reactivation during the folding of alkaline phosphatase. 998 86

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.
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PMID:Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond. 1066 87

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.
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PMID:Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein. 1103 44

Our initial studies of hydrogen-deuterium (H-D) exchange of tryptophan 109 in Escherichia coli alkaline phosphatase (AP) suggested that significant local unfolding of the protein might occur to allow for the exchange reaction, which is very slow at room temperature (Fischer et al., Biochemistry 39 (2000) 1455-1461). In order to investigate whether the partial unfolding and/or 'breathing' motions leading to H-D exchange were part of the unfolding pathway of the protein we prepared a series of mutants, designed to produce cavities around the exchanging residue, and compared their rates of H-D exchange to their lability (rate of inactivation) in guanidine hydrochloride (Gd:HCl). The complex unfolding kinetics of the mutants in the presence of Gd:HCl showed several components with rates that differed substantially among these proteins, but none of the rates of denaturation induced with Gd:HCl was consistently correlated with the H-D exchange rates. We conclude that the partial opening of the AP structure during the H-D exchange of tryptophan 109, although very slow, is not a rate determining step in the unfolding of this protein.
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PMID:Differences in the pathways for unfolding and hydrogen exchange among mutants of Escherichia coli alkaline phosphatase. 1134 35

The in vitro reactivation of unfolded Escherichia coli alkaline phosphatase (AP) in the presence of the two natively bound metals Zn2+ and Mg2+ produces two protein species, characterized by different guanidine hydrochloride denaturation kinetics. The high-lability AP form slowly converts to the low-lability form in a first-order reaction with a characteristic lifetime (inverse rate constant) of approximately 300 h at pH 8.0 and 25 degrees C. Addition of Zn2+ and Mg2+ ligands to (folded) apo-AP also produces two protein species, with denaturation kinetics and a long conversion lifetime similar to those found in refolding AP. In contrast, adding Zn2+ alone to apo-AP produces only the high-lability species with no subsequent structural change, suggesting that Mg2+ binding is the event which is responsible for the production of the low-lability AP. The rate of conversion from high- to low-lability AP was found to be linearly dependent on Mg2+ concentration, indicating that Mg2+ binding is rate limiting for this reaction. Experiments where either Zn2+ or Mg2+ was added first, with the second metal added later, show that Mg2+ binding is slowed by the prior presence of bound Zn2+. Mg2+ binding to Zn-AP also slightly increases the enzymatic activity; however, the extent of formation of the low-lability species is related to the square of the Mg2+-induced activity increase. Thus the binding of two Mg2+ to AP produces the dramatic reduction in the rate of denaturation that characterizes the low-lability species. The data suggest the possibility of long distance intersubunit interactions and a role for Mg2+ in providing "kinetic stability" for AP.
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PMID:Mg2+ binding to alkaline phosphatase correlates with slow changes in protein lability. 1155 Dec 21


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