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)

Immunophenotype analysis of 17 childhood medulloblastoma (MED) and supratentorial primitive neuroectodermal tumors (SPNET) was performed on frozen sections using 16 monoclonal antibodies (MoAb) with the biotin-streptavidin alkaline phosphatase immunohistochemical technique. Neuroectodermal associated antigens, reacting with MoAb UJ13/A, UJ127.11, UJ167.11, and UJ223.8 were detected on greater than 10% of the cells in 15 of 17 MED/SPNET. Thy-1 was present on 14 of 17 tumors and absent on two of three SPNET. Neuronal (NF) and glial (GFAP) differentiation markers were evaluated. NF-H was demonstrated in 15 of 17, NF-M in six of 17 and NF-L in one of 17 tumors; GFAP was positive in nine of 17 patients. In nine of 17 MED/SPNET both proteins were present within the same tumor. Common leukocyte antigen was demonstrated on greater than 50% of the cells in four of 14 tumors as were shared tumor/leukocyte markers using monoclonal antibodies Thy-1, PI153/3, UJ308. The most frequent MED immunophenotype analysis was UJ 13/A+, UJ 127.11+, UJ 167.11+, UJ223.8+, PI 153/3+, A2B5+, GFAP+, NF-H+, and CLA-, NF-M-, NF-L-, 215-, 275-, 282.1-. The authors conclude that MED and SPNET are heterogeneous for expression of 16 markers and have similar immunophenotype analysis profiles, supporting the concept of their common, neuroectodermal origin. Common leukocyte antigen on both tumor cells and leukocytes precludes identification of tumor infiltrating leukocytes using monostaining techniques.
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PMID:Immunophenotype profile of childhood medulloblastomas and supratentorial primitive neuroectodermal tumors using 16 monoclonal antibodies. 219 9

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.
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PMID:Two monoclonal antibodies recognize Alzheimer's neurofibrillary tangles, neurofilament, and microtubule-associated proteins. 243 80

The phosphorylation state of human and bovine spinal cord neurofilaments (NF) was studied by direct phosphate analysis and carbocyanine dye ("Stains-all") binding to NF polypeptides resolved on SDS-polyacrylamide gels. Electrophoretically purified NF-H (200 kDa), NF-M (160 kDa), and NF-L (68 kDa) of human origin contained 24, 18, and 4 mol phosphate/mol protein, whereas bovine NF contained 53, 23, and 5 mol phosphate/mol protein, respectively. Incubation of NF preparations with E. coli alkaline phosphatase removed about 55% of the phosphate from NF-H, about 30% of the phosphate from both human and bovine NF-M, but did not change the phosphate content of NF-L. This treatment also inhibited or substantially reduced the binding of electroblotted NF-H and NF-M to 2 anti-NF monoclonal antibodies known to recognize phosphorylated sites on projection side arms. "Stains-all" was found to be a very sensitive probe for detection of phosphorylated cytoskeletal proteins. Without the phosphatase treatment, NF and other phosphoproteins, MAP1, MAP2, tubulin, and tau, all bound the carbocyanine dye on SDS gels, forming blue dye-protein complexes. Measured densitometrically at 615 nm, the staining intensity (relative units/mol protein) was 9, 9, and 3 for human and 10, 13, and 6 for bovine NF-H, NF-M, and NF-L, respectively. NF-H bound the dye less efficiently than was expected from its phosphate content. After phosphatase treatment, NF-H, with half of its phosphate residues remaining, no longer formed blue complex with "Stains-all," the staining intensity of NF-M decreased by 20-40%, and the staining of NF-L was not changed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphatase and carbocyanine dye binding define different types of phosphate groups in mammalian neurofilaments. 244 39

We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [35S]methionine or [32P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [35S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6-18 h for NF-M, 18-24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [32P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195-210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non-phosphorylated neurofilaments to predominantly phosphorylated ones within a region of the axon between 200 and 1000 microns downstream from the cell body. These experiments demonstrate that the addition of most phosphate groups to NF-M and NF-H takes place within a proximal region of the axon. The rapid appearance of modified forms of NF-L after synthesis may imply that processing of this subunit occurs at least partly in the cell body. The presence of a substantial pool of Triton-insoluble, unmodified subunits early after synthesis indicates that the heaviest incorporation of phosphate occurs after neurofilament proteins are polymerized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport. 246 28

The low (NF-L) and middle (NF-M) molecular weight (Mr) neurofilament (NF) subunits are expressed before the high (NF-H) Mr NF subunit in embryonic neurons. Thereafter, NF-M attains its mature state of phosphorylation more rapidly than does NF-H. However, little is known about NF subunit expression during cell division. A rapidly dividing medulloblastoma cell line (D283 MED), therefore, was examined using flow cytometry, immunochemistry, and a large panel of NF subunit-specific polyclonal and monoclonal antibodies. Many of the monoclonal antibodies (MAbs) distinguished NF-H and NF-M in different states of phosphorylation. By flow cytometry, more than 90% of the D283 cells expressed NF-H and NF-M in different states of phosphorylation, and an antiserum specific for the carboxy terminus of NF-L labeled more than 60% of these cells. Furthermore, the fluorescence intensity produced by MAbs that detected phosphorylated versus nonphosphorylated NF-H and/or NF-M epitopes, appropriately decreased or increased, respectively, by preincubating the D283 cells with alkaline phosphatase. In contrast, cell staining with antibodies specific for phosphate-independent NF protein epitopes did not change substantially as a result of enzymatic dephosphorylation. These results agreed closely with those obtained from studies of normal human spinal cord NF extracts. However, NF-H, NF-M, and NF-L were expressed throughout the cell cycle in dual parameter studies of D283 cells labeled with an antibody and propidium iodide. Nevertheless, reductions in the fluorescence intensity produced with most of these antibodies late in the cell cycle suggested that NF proteins may be subject to modifications in their structure or accessibility to antibody probes during different phases of the cell cycle. These data led to the conclusion that NF subunits are expressed throughout the cell cycle in cultured human medulloblastoma cells, but that subtle changes in the immunoreactivity of these proteins occur during cell division.
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PMID:Phosphate-dependent and independent neurofilament protein epitopes are expressed throughout the cell cycle in human medulloblastoma (D283 MED) cells. 247 22

Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988). Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections. We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy. (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing. This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H. Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples. (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy. No difference in the density was noted between control and dephosphorylated NFs. (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy. The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E. coli alkaline phosphatase. These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro.
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PMID:The effects of dephosphorylation on the structure of the projections of neurofilament. 253 87

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.
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PMID:Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH. 255 34

The degradation of phosphorylated and dephosphorylated neurofilament proteins by the Ca2+-activated neutral proteinase calpain was studied. Neurofilaments were isolated from bovine spinal cord, dephosphorylated by alkaline phosphatase (from Escherichia coli) and radioiodinated with [125I]-Bolton-Hunter reagent. The radioiodinated neurofilament proteins (untreated and dephosphorylated) were incubated in the presence and absence of calpain from rabbit skeletal muscle, and the degradation rates of large (NF-H), mid-sized (NF-M) and small (NF-L) neurofilament polypeptides were analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. The degradation of dephosphorylated neurofilament proteins occurred at a higher rate, and to a greater extent, than did that of the phosphorylated (untreated) neurofilament proteins. The dephosphorylated high-molecular-mass neurofilament (NF-HD) was proteolyzed 6 times more quickly than the untreated NF-H. The degradation rate of the NF-M and NF-L neurofilament proteins was also enhanced after dephosphorylation, but less than that of NF-H. This indicates that the dephosphorylation of neurofilament proteins can increase their sensitivity to calpain degradation.
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PMID:Dephosphorylation of neurofilament proteins enhances their susceptibility to degradation by calpain. 285 97

A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF proteins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immunochemically distinguish and characterize distinct and differentially phosphorylated isoforms of NF subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific for NF-M detected their specific NF subunit regardless of whether or not the NFs had been treated with alkaline phosphatase, and such antibodies were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized epitopes in the COOH termini of these subunits. Significantly, the latter mAbs could discriminate different isoforms of NF-M and NF-H, depending on the phosphorylation state of each variant. Such mAbs were assigned to one of 4 distinct categories on the basis of their performance in immunoblots of progressively dephosphorylated rat NF samples and by immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs preferentially stained neuronal perikarya and dendrites, and they recognized only extensively dephosphorylated (and nonphosphorylated) NF-H; (2) P[+] mAbs stained axons more strongly than perikarya, and primarily blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M; (3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in blots recognized only the extensively phosphorylated forms of NF-H and NF-M (i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ] mAbs also predominantly stained axons, but the briefest alkaline phosphatase treatment abolished the NF-M and NF-H immunobands produced by these mAbs. Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo. This provided additional evidence that mAbs can detect all 4 phosphorylation-dependent endogenous isoelectric variants of NF-H and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats. 311 89

Exhaustive in vitro dephosphorylation of porcine neurofilaments (NFs) by alkaline or acid phosphatase did not cause a dissociation of the 210-kD (NF-H), 160-kD (NF-M), or 70-kD (NF-L) subunits and had no effect on the reassembly of NFs from urea or guanidine solution. Electron microscopy revealed that the NFs reassembled from isolated or dephosphorylated subunits had similar morphologies. Phosphatase treatment caused significant increases in the mobilities of NF-M and NF-H on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the subunits underwent marked conformational changes after dephosphorylation. Chemical phosphate analysis showed that as isolated NF-H, NF-M, and NF-L contained about 22, 11, and 3 mol phosphate/mol polypeptide, respectively. The corresponding values for the three subunits from alkaline phosphatase-treated NFs were about 8, 6, and 2 mol phosphate/mol polypeptide, respectively. These results indicate the occurrence of a class of phosphate moieties that is not accessible to exogenous phosphatases.
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PMID:Dephosphorylation of neurofilaments by exogenous phosphatases has no effect on reassembly of subunits. 373 90


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