Evidence That the Synaptic Phosphoprotein B-50 Is Localized Exclusively in Nerve Tissue

The localization of the phosphoprotein B-50 (molecular weight 48,000 isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B-50 protein. Two-dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and in immunochemical detection method employing an anti B-50 antiserum revealed the presence of B-50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B-50 in nervous tissue. In addition, the regional distribution of endogenous B-50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B-50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1-24 is discussed.     

Calcified Microspheres as Biological Entities and Their Isolation from Bone

Calcified microspheres, about 1µm in diameter, appear at sites of bone formation where they invest the collagenous matrix, become confluent and disappear. Evidence that the particle boundaries are not lost with compaction but merely deformed is supported in section by the granular histochemical staining of the inorganic phase for bone salt, lipid, fibronectin and acid phosphatase in osteomalacic, acid-etched and normal human bone. Their persistence as discrete objects is confirmed by the application of methods for their isolation from the collagenous matrix of immature mouse calvarium and mature bovine femur. Five methods have been used to extract them and include (i) biochemical, (ii) chemical, (iii) mechanical, (iv) pyrogenous and (v) biological separation. Under the optical microscope, all isolates consisted of similar discrete objects and bridged assemblies, whose birefringence varied with treatment. After decalcification, their organic ghosts remained. Each isolated microsphere had a complex substructure of clusters of non-collagenous calcified filaments surrounding a less dense centre. The filaments were 5nm in diameter with a 5nm periodicity and regular fine interfilamentous connections. It is concluded that the microspheres are independent, complex, pervasive and central to the containment (i.e. packaging) of calcium phosphate in bone. Their extraction will enable further analysis.     

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.Phosphorylation of proteins is an integral part of the signal transduction of eukaryotic cells as it modulates the activity of complex protein networks. Although Western blot- and immunoprecipitation-based MS approaches (1, 2) can lead to detailed insights into these processes, most of the integrated approaches only allow a static view of protein phosphorylation because they are not suitable for the screening of hundreds of samples. Either planar or bead array-based sandwich immunoassays can be used to analyze the quantity and activation state of signaling molecules in multiplex, enabling the systematic profiling of protein abundance and post-translational modifications (3–6) in hundreds of samples. However, multiplex immunoassays are only suitable for the simultaneous analysis of a limited number of proteins. The detection of comprehensive phosphorylation patterns is difficult as this involves assay systems that are incompatible with multiplexing.In principle, two sandwich immunoassay setups are possible for probing the phosphorylation state of a protein. The first setup applies a capture antibody specific for a non-modified part of the protein and uses a phosphorylation state-specific detection antibody. When applied to an array-based format, however, this setup does not allow for the simultaneous measurement of the abundance and the degree of phosphorylation (3, 4). A mixture of detection antibodies, one specific for the phosphorylation site and one specific for the non-modified site of the protein, would bind simultaneously to the two different epitopes, and assay signals could not be further deconvoluted by the spatial or color code of the array. The second sandwich immunoassay setup for the analysis of protein phosphorylation applies a phosphorylation state-specific capture antibody and a protein-specific detection antibody. In such a setup, an anti-phosphotyrosine antibody (e.g. mAb 4G10) cannot be applied as a capture antibody because a huge variety of tyrosine phosphorylated proteins would be captured, and specific signals could rarely be deconvoluted. Using capture antibodies that bind to phosphorylated epitopes in the context of their flanking amino acids is not a problem until a multiplex readout is desired. If one antibody specific for the phosphosite and one antibody specific for the abundance of a protein are used together in a multiplex assay panel they might compete for their analyte. The situation becomes even more complex if the protein of interest contains various phosphorylation sites such as e.g. the epidermal growth factor receptor. Several capture antibodies target different epitopes of the same protein and therefore compete for the overall amount of targeted protein in the sample, thus making a valid simultaneous measurement problematic.Although different ways of tackling the problem of assay multiplexing are in use, we demonstrated the feasibility to sequentially perform such incompatible assays from the same sample using a magnetic particle handler that moves particles through the samples and reagents (Fig. 1). Using a model assay, we confirmed that suspension bead array-based immunoassays work under ambient analyte conditions. As described by Roger Ekins (7), decreasing of the amount of capture antibody in a sandwich immunoassay setup from a macrospot (e.g. a microtiter plate assay) to a microspot generates a scenario where only a tiny fraction of the present target analytes is captured on the microspot. Therefore, the overall concentration of the analyte molecules in the sample does not change significantly even in the case of low target concentrations and high affinity binding reactions. Furthermore, as the initial concentration of the analyte is not significantly changed when performing a miniaturized sandwich immunoassay, multiple post-translational modifications within the same protein can be measured either in sequence or in parallel in the same multiplex panel.Open in a separate windowFig. 1.Sequential multiplex analyte capturing. Magnetic suspension bead array assays can be performed sequentially, reusing the same sample material (indicated by the blue arrow). The use of a magnetic particle handler enables the quantitative transfer (black arrow) of the magnetic beads from the sample well into the wells containing washing solutions or other assay reagents. Magnetic beads from the first bead array panel are incubated with the samples to capture their respective analyte. Then the magnetic beads are subjected to washing and detection steps and are finally transferred into the readout plate (first row). After retracting the magnetic suspension bead array of the first assay panel from the sample, a bead array from the second assay panel is added and processed as described above but using different detection antibodies (second row). A third bead array assay panel can be applied after removing the second panel (third row) and so on.By probing tumor cell lines for the abundance of seven different receptor tyrosine kinases and their generic tyrosine phosphorylation, we generated complex phosphorylation patterns and thereby demonstrated the potential of this approach. More importantly, demonstrating ambient analyte conditions allowed the parallel detection of phosphorylation at different sites of the EGFR1 using phosphorylation site-specific antibodies as capture molecules with one assay panel. Phosphorylation of eight different sites and the abundance of the EGFR could be quantified relative to one another without any interference of the different immunoassays during multiplexing because competition for the analyte can be prevented by running the assays under ambient analyte conditions.     

Resolution of Rat Brain Synaptic Phosphoprotein B-50 into Multiple Forms by Two-Dimensional Electrophoresis: Evidence for Multisite Phosphorylation

Phosphoprotein B-50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat-bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5-5.0). As visualized by two-dimensional gel electrophoresis, B-50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B-50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B-50 was in part due to a glycoprotein nature of B-50 was studied extensively. However, none of the six different methods used revealed the presence of glyco-moieties in B-50.     

New Evidence for a Discrete Supratemporal Bone in the Jurassic Ichthyosaur Temnodontosaurus

The temporal region in Temnodontosaurus trigonodon and Temnodontosaurus platyodon consists of three discrete ossifications. These bones are identified as the supratemporal, bordering much of the temporal opening, and in mid-cheek, the squamosal, which is in contact with the quadratojugal along its ventral margin. Interpretation of the bone in mid-cheek position as a neomorphic "supernumerary" ossification is rejected. The supratemporal bone of ichthyosaurs is functionally convergent with the squamosal of diapsids. The size and configuration of the supratemporal casts doubt on the supposition that ichthyosaurs were diapsids.     

酵母细胞分裂周期突变株中磷酸化蛋白的变化

最近对酵母细胞腺苷酸环化酶和依赖于cAMP的蛋白激酶基因的分子生物学研究,说明磷酸化蛋白在细胞周期中有重要作用。本文用聚丙烯酰胺凝胶电泳研究了酵母细胞分裂周期突变株中蛋白质的磷酸化。依赖cAMP的突变株AM18生长在无cAMP的培基中时,发生了几种磷酸化蛋白的变化,最明显的是分子量为72K Da的蛋白的积聚。细胞分裂周期温度敏感突变株cdc35生长在高于允许温度对,以及野生株生长在稳定期时,也出现类似现象。在这三种情况下,72K Da磷酸化蛋白同时还具有相同的pI值(pI=4.7),磷酸化都发生在苏氨酸残基上,用蛋白酶部分水解法证明它们有相似的肽谱。这些结果说明它们为同一蛋白。     

Studies on Phosphoprotein Phosphatase in Chick Embryo

Several properties of partially purified phosphoprotein phosphatase from chick embryo are described. The enzyme was active toward casein, phosvitin and phosphopeptone, but not toward low molecular weight phosphate esters including aliphatic and aromatic phos-phomonoesters, a phosphodiester, pyrophosphates and phosphoamides. The enzyme was not activated by reducing compounds. Heavy metal ions and sulfhydryl inhibitors inhibited the enzyme activity, but the inhibited enzyme was partially reactivated with cysteine. Metal chelating agents also inhibited the activity. To the oxalate treated enzyme, Fe⁺⁺ and Co⁺⁺ had a stimulatory effect. Differences in property between phosphoprotein phosphatases of chick embryo and of mammalian tissues are discussed.     

Phosphoprotein phosphatase activity in the thyroid.

Phosphoprotein phosphatase activity in the calf thyroid was found in various subcellular fractions. The relative amount in each fraction varied according to the substrate used: The 500g fraction had the highest specific activity when protamine was used, while the 5000g fraction was highest when histone was used. Triton X-100 tended to increase activity in all the particulate fractions, the greatest change being found in the 105,000g pellet. DEAE chromatography of the 105,000 g supernatant resolved at least three peaks of phosphoprotein phosphatase activity.     

Secreted Phosphoprotein 24 kD (Spp24) and Spp14 Affect TGF-β Induced Bone Formation Differently

Transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) have opposing but complementary functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore, regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal truncation products all bind to TGF-β1 and TGF-β2 with a similar but somewhat less affinity than they bind BMP-2; that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-β with greater affinity than do the truncation products; that FL-Spp24 inhibits TGF-β2 induced bone formation in vivo, but Spp14.5 does not; and that co-administration of FL-Spp24 or Spp14.5 with TGF-β2 in vivo is associated with a reduction in the amount of cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-dose TGF-β administration in vivo is associated with greater bone formation than high-dose TGF-β administration, and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-β activity or availability during bone growth and development. The similarities and differences of the interactions between Spp24 proteins and TGF-β compared to the interaction of the Spp24 proteins and BMPs have significant implications with respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal disorders.     

Phosphoprotein Component of Vaccinia Virions

The recent discovery of a protein kinase activity in vaccinia virions led us to search for a viral protein which is phosphorylated in vivo. Vaccinia virus was radioactively labeled by infecting cells in the presence of (32)P(1). A phosphoprotein was isolated from purified delipidated virions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phosphoprotein appeared to be a specific viral component induced after infection. More than 60% of the phosphoprotein was associated with viral cores. The electrophoretic mobility of the protein suggested that it has a molecular weight of 11,000 to 12,000. Phosphoserine was liberated by acid hydrolysis and identified by electrophoresis with known standards. Tryptic digests of the purified phosphoprotein were analyzed by two-dimensional electrophoresis and chromatography on thin-layer cellulose plates, and a single major phosphopeptide was resolved. The high selectivity of phosphorylation suggested that the process has a specific function.     

Two-Dimensional Immobilized Metal Affinity Electrophoresis for Capturing a Phosphoprotein

A two-dimensional immobilized metal affinity electrophoresis method is described here. In this method, ferric ions are immobilized in the second-dimensional polyacrylamide gel to extract the phosphoprotein β-casein from a mixture containing proteins with a broad range of pI and MW. Native 7.5–15% gradient tris-glycine gel with SDS tris-glycine gel running buffer are used so that proteins can be separated according to their molecular mass in the second dimension.     

Phosphoprotein Phosphatase Activities in Alzheimer Disease Brain

Abstract: Microtubule-associated protein τ is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of τ to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated τ could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of τ might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used ³²P-labeled phosphorylase kinase and poly(Glu.Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/ phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of τ in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of β-amyloid by augmenting the amyloidogenic pathway processing of β-amyloid precursor protein.     

Phosphoprotein phosphatase nonspecifically hydrolyzes CoA

CoA hydrolysis was studied by a homogenous phosphoprotein phosphatase (EC 3.1 3.16) preparation from bovine spleen nuclei at pH 5.8. Phosphoprotein phosphatase catalyzed hydrolysis of the CoA 3'-phosphoester bond to form dephospho-CoA and Pi. The Km value for phosphoprotein phosphatase with CoA as substrate was 3.7 mM, the specific activity - 0.26 mmol Pi.min-1.mg-1. Phosphoprotein phosphatase did not essentially catalyze the calcium pantothenate hydrolysis (not more than 2% as compared with the CoA hydrolysis rate).     

Modular Organization of Rabies Virus Phosphoprotein

A phosphoprotein (P) is found in all viruses of the Mononegavirales order. These proteins form homo-oligomers, fulfil similar roles in the replication cycles of the various viruses, but differ in their length and oligomerization state. Sequence alignments reveal no sequence similarity among proteins from viruses belonging to the same family. Sequence analysis and experimental data show that phosphoproteins from viruses of the Paramyxoviridae contain structured domains alternating with intrinsically disordered regions. Here, we used predictions of disorder of secondary structure, and an analysis of sequence conservation to predict the domain organization of the phosphoprotein from Sendai virus, vesicular stomatitis virus (VSV) and rabies virus (RV P). We devised a new procedure for combining the results from multiple prediction methods and locating the boundaries between disordered regions and structured domains. To validate the proposed modular organization predicted for RV P and to confirm that the putative structured domains correspond to autonomous folding units, we used two-hybrid and biochemical approaches to characterize the properties of several fragments of RV P. We found that both central and C-terminal domains can fold in isolation, that the central domain is the oligomerization domain, and that the C-terminal domain binds to nucleocapsids. Our results suggest a conserved organization of P proteins in the Rhabdoviridae family in concatenated functional domains resembling that of the P proteins in the Paramyxoviridae family.     

A Radioimmunoassay for the Phosphoprotein B-50: Distribution in Rat Brain

A radioimmunoassay (RIA) for the B-50 protein was developed to determine B-50 in total homogenates of rat tissues. A tracer of purified B-50 was prepared at high activity (10-30 microCi/micrograms protein) by phosphorylating B-50 with carrier-free [gamma-32P]ATP, catalyzed by purified protein kinase C. The RIA was performed using affinity-purified anti-B-50 immunoglobulins G in a detergent containing medium and detected B-50 at levels of 0.1-10 ng. Specificity of the antibodies was ascertained by immunoprecipitation of B-50 from a crude mitochondrial membrane fraction from rat brain and by immunoblotting. For the B-50 content in rat brain the following distribution pattern was found: medulla spinalis less than cerebellum less than hippocampus; cerebral cortex less than periaqueductal gray less than septum. The septum contained 80 micrograms/g tissue weight. The level in liver homogenates was below detection. The regional distribution is in fair agreement with the pattern of the endogenous B-50 phosphorylation in rat brain synaptosomal plasma membranes previously reported.