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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently shown that the major proteins from bovine seminal plasma, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins) are novel phospholipid-binding proteins. These proteins show a Ca(2+)-independent interaction with phosphorylcholine (PrC) containing lipids and this group acts as a specific binding site for BSP proteins on the sperm membrane. In this study, we investigated the biochemical basis of the binding of BSP proteins to the phosphorylcholine moiety using different affinity chromatography matrices containing active groups analogous to PrC. Proteins from bovine seminal fluid were applied to a column containing p-aminophenyl phosphorylcholine coupled to Agarose (PPC-agarose). Unadsorbed proteins were washed out with buffer (Tris-HCl) and elution of bound proteins was assessed with the same buffer containing different eluting agents. The adsorbed proteins were eluted with buffer containing specific (PrC or choline chloride), denaturing agent (urea), or chaotropic agent (sodium thiocyanate) and identified as BSP proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. At high ionic strength, these proteins could also be adsorbed on quaternary methylamine coupled to silica and diethylaminoethyl coupled to Sephadex and were eluted specifically with phosphorylcholine, choline chloride, or urea. These data suggest a structure-dependent hydrophobic interaction of the ligands with BSP proteins. Furthermore, binding experiments using equilibrium dialysis indicated that there are two choline binding sites per mole of protein. In addition, the combination of affinity chromatography columns and linear gradient of eluting agents allowed the complete fractionation of the different BSP proteins. The availability of an affinity chromatography method should aid in those studies aimed toward the understanding of the physiology of these phospholipid-binding proteins.
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PMID:Interaction of a novel class of phospholipid-binding proteins of bovine seminal fluid with different affinity matrices. 837 72

Bone acidic glycoprotein-75 (BAG-75) displays a strong propensity to self-associate to form large fibrillar complexes above concentrations of 7 x 10(-8) M; acidic phosphoproteins osteopontin and bone sialoprotein do not form similar complexes. Although the majority of the data supporting this conclusion is derived from in vitro studies, the fact that similar sized complexes are observed in crude extracts of bone and calcified cartilage suggests that macromolecular BAG-75 complexes are also a component of mineralized matrices in vivo. An awareness of the existence of complexes in extracts from bone necessitates that these forms are accounted for in terms of the relative amounts of individual acidic phosphoproteins in bone matrix. We now estimate that the amount of BAG-75 in rat calvarial bone is equivalent to that of osteopontin. While BAG-75 is capable of binding up to 139 atoms of calcium/mole with an average affinity constant of 0.5-1.0 x 10(-3) M, millimolar concentrations of calcium are not required for self-association. Assuming macromolecular diffusion within osteoid is restricted, osteoblastic cells could control the extent of self-association through the rate at which BAG-75 is synthesized and secreted into the osteoid matrix. Based on these findings, we hypothesize that BAG-75 self-associates to form fibrillar complexes in vivo which function in a supportive mechanical role and/or as an electronegative ionic barrier. Electronegative BAG-75 barrier structures could play a role in concentrating phosphate ions within bone matrix, thus facilitating mineralization.
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PMID:Bone acidic glycoprotein-75 self-associates to form large macromolecular complexes. 908 51

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.
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PMID:Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins. 908 59

Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear + plasma membrane, cytosolic and microsomal, and used in in vitro phosphorylation of purified 20 kDa bovine amelogenin in the presence of 32P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca2+ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca2+ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca2+, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly CaZ+ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of 14C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, 14C-mercaptoethanol, 0.64 moles 14C-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by 14C-mercaptoethanol, providing 0.46 and 0.88 moles 14C-incorporated/mole respectively. Further studies of the 14C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.
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PMID:Enamel specific protein kinases and state of phosphorylation of purified amelogenins. 1106 30

Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.
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PMID:Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts. 3005 20