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)

New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca- and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5'-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H2O2 which decomposes at 55 degrees-60 degrees C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H2O2 mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H2O2-DAB and especially the H2O2-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.
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PMID:The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure. New methods for light microscopic phosphatase histochemistry and immunohistochemistry. 285 63

A monoclonal antibody specific for cardiac troponin T has been used to investigate troponin changes during development in the rat heart. Specificity of the antibody was determined by immunoblot analysis with purified bovine cardiac troponin. In the rat heart, immunoblot analysis shows that anticardiac troponin T reacts with a 42.5-kDa band in fetal ventricles and with a 41-kDa band in adult ventricles. The faster migrating troponin T is present in traces in the fetal heart and increases markedly during the first 2 weeks after birth, concomitantly with the progressive decrease of the slower migrating form that is no longer detectable in the adult. The pattern of reactivity of the monoclonal antibody is not modified by alkaline phosphatase pretreatment, suggesting that the antibody is not specific for a phosphorylated epitope. Conditions known to affect cardiac myosin composition, such as hypothyroidism and hypertrophy secondary to systemic hypertension, do not change the troponin T isoform profile of adult rat ventricles. The expression and accumulation of the adult isoforms of troponin T are not suppressed by propylthiouracil treatment of pregnant and nursing rats.
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PMID:Troponin T switching in the developing rat heart. 297 62

The specificity of cytosolic protein phosphotyrosine (PPT) phosphatases was investigated using different peptides and proteins that were phosphorylated on tyrosine residues by the EGF receptor kinase. The acidic phosphoproteins, serum albumin, casein, and myosin light chains, were dephosphorylated by the PPT phosphatases with apparent Km values of 1.2 to 12.5 microM and apparent velocities of 0.2 to 18 mumol/min/mg. In contrast, [Tyr(32P)]histone and the phosphotyrosine peptides [Val5]angiotensin and RR-src, a peptide with sequence Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, were unreactive with the PPT phosphatases. However, each of these unreactive phosphopolypeptides was dephosphorylated under the same conditions by calf-intestine alkaline phosphatase. The data reveal how PPT phosphatase activity has been ascribed to different cellular enzymes. When acidic phosphotyrosine proteins were used as substrates in assays for PPT phosphatase activity the cytosolic enzymes were isolated, whereas when phosphotyrosine histones were used as substrates only the membrane-bound alkaline phosphatase was detected. Apparently the protein tyrosine kinase and the protein tyrosine phosphatases do not have the same specificity, so substrates such as histone, angiotensin, or RR-src are phosphorylated but not hydrolyzed. Therefore, these polypeptides would be ideal for the characterization of protein tyrosine kinases in cellular extracts.
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PMID:Specificity of protein phosphotyrosine phosphatases. Comparison with mammalian alkaline phosphatase using polypeptide substrates. 298 3

We performed in situ hybridization of myosin heavy-chain (MHC) mRNA on rabbit muscle using a biotin-labeled complementary RNA probe. An 1107-nucleotide fragment from an alpha-cardiac MHC cDNA was used to transcribe an RNA probe 97% similar to slow-twitch and 75% similar to fast-twitch sequences. Serial sections were used to identify slow-twitch fibers in medial gastrocnemius, soleus, and tibialis anterior by immunofluorescence of slow MHC and oxidative capacity by histochemistry. Slow-twitch fibers hybridized by the RNA probe stained heavily after detection with streptavidin-alkaline phosphatase (89% dark and 11% medium density). Fast-oxidative fibers stained intermediately (26% dark, 58% medium, and 16% light) and fast-glycolytic fibers stained lightly (12% medium and 88% light). Biotin-labeled probe and enzymatic detection allowed greater resolution of the subcellular location of the MHC mRNA, a distinct advantage over isotope labeling and autoradiography. A non-uniform distribution of MHC mRNA was recognized within an adult skeletal muscle fiber. High concentrations of MHC mRNA were found under the sarcolemma and between the myofibrils, suggesting the existence of a distribution mechanism. The combination of in situ hybridization and immunocytochemistry allows rapid subcellular localization of both MHC mRNA and its translated protein.
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PMID:In situ hybridization and immunocytochemistry in serial sections of rabbit skeletal muscle to detect myosin expression. 305 72

Six polypeptides resolved by two-dimensional electrophoresis of homogenates from human skeletal muscle have been identified as tropomyosin by electrophoretic and immunochemical methods. The 6 proteins are consistently present in approximately the same abundance in normal biceps, deltoid, gastrocnemius, and quadriceps muscle. Analysis of samples from individuals with Becker's dystrophy, Duchenne dystrophy, limb girdle dystrophy, polymyositis, myopathy related to vitamin E deficiency, type II fiber deficiency, and from an infant with indistinct fiber type differentiation, however, showed quantitative variations in the tropomyosin pattern. Muscle with histochemically demonstrated type II fiber deficiency lacked two of the normal tropomyosin proteins and the type II myosin light chains. Muscles lacking type I myosin light chains were deficient in a different pair of tropomyosin proteins. The results suggest that normal human skeletal muscle contains one major type of tropomyosin protein (beta-tropomyosin) common to both fast and slow fibers, together with two other major proteins (alpha-tropomyosin and alpha'-tropomyosin), one of which is specific to fast fibers and the other to slow fibers. Preliminary data from the reaction of muscle homogenates with alkaline phosphatase indicate that 3 of the 6 tropomyosin polypeptides resolved by two-dimensional electrophoresis are phosphorylated forms of the alpha-, alpha'-, and beta-tropomyosins.
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PMID:Heterogeneity of human skeletal muscle tropomyosin. 389

Myosin purified from a murine myeloid leukaemia cell line (M1) that had been incubated with [32P]orthophosphate incorporated 32P into the heavy, but not the light, chain. When the heavy chain was dephosphorylated by bacterial alkaline phosphatase, myosin that had low actin-activated ATPase activity gained higher activity only in the presence of the light-chain kinase. In the absence of the light-chain kinase, however, the Mg2+-stimulated ATPase activity of myosin was not activated by actin, regardless of phosphatase treatment. These results indicate that the activity of M1 myosin ATPase is regulated by phosphorylation of both the light and heavy chains. A scheme for this regulation by phosphorylation is presented and discussed.
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PMID:Phosphorylation of the myosin heavy chain. Its effect on actin-activated Mg2+-stimulated ATPase in leukaemic myeloblasts. 613 30

6-Tridecylresorcylic acid (TRA) isolated from a primula Lysimachia japonica Thunb. inhibited contraction of myofibrils, superprecipitation of myosin B, and ATPase activities of myosin and actomyosin prepared from rabbit skeletal muscle in a dose-dependent manner. The IC50 values in molarity of TRA were as follows: myosin (K+,EDTA)-ATPase, 3.5 X 10(-6); myosin Ca2+-ATPase, 3.5 X 10(-5); and actomyosin Mg2+-ATPase, 1.6 X 10(-5). The inhibition of ATPase activity of myofibrils by TRA was virtually reversed by washing with the fresh saline solution. Kinetic analysis of inhibitory effects of TRA suggests that the inhibition of (K+,EDTA)-ATPase activity of myosin or subfragment-1 is parabolic noncompetitive. TRA had no effect on alkaline phosphatase and choline acetyltransferase activities. TRA may provide a useful chemical tool for the study of the molecular mechanisms of actin-myosin contractile systems.
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PMID:6-Tridecylresorcylic acid, a novel ATPase inhibitor that blocks the contractile apparatus of skeletal muscle proteins. 623 63

Matrix vesicles, extracellular microstructures known to be involved in endochondral calcification, are rich in alkaline phosphatase and have been shown to contain actin. The mechanism of matrix vesicle formation in chondrocytes is not well understood. Chondrocytes from the epiphyseal growth plate, when grown in primary culture, elaborate alkaline phosphatase-rich vesicles. We examined the distribution of the cytoskeletal proteins actin, myosin, tubulin, and vinculin at various time-points during culture using indirect immunofluorescent labeling. Concomitantly, the production of alkaline phosphatase-containing matrix vesicles was also followed. Cell morphology changed noticeably at two distinct stages during the 22-day culture period: Immediately after release from the growth plate the cells were rounded, but after 4 days of culture they began to spread out and acquire irregular shapes with distinct filopodia. By 13 days, as the cells attained confluency, they reacquired a rounded, polygonal appearance. At all time-points, tubulin was seen as a dense network of microtubules radiating from the perinuclear region throughout the cytoplasm toward the cell periphery. Initially actin was seen in filamentous form, but displayed a punctate distribution focused at contact points during the cell-spreading stage of culture. After confluency, actin was concentrated at cell-cell junctions. Initially, vinculin was diffusely distributed, but became focused in multiple adhesion plaques and at the termini of filopodia during the cell-spreading stage of culture. Following confluency vinculin became concentrated at cell-cell junctions. Myosin was observed at all time-points in small, intensely localized focal points in the cytoplasmic region of the cells and was consistently absent from the nuclear and peripheral regions. The amount of myosin in the cells increased steadily with time in culture. Elaboration of alkaline phosphatase-rich vesicles, which corresponded closely with the rounded morphology of early and late stages of culture, may be correlated with contact inhibition.
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PMID:Correlation between distribution of cytoskeletal proteins and release of alkaline phosphatase-rich vesicles by epiphyseal chondrocytes in primary culture. 642 67

Dictyostelium myosin is composed of two heavy chains and two pairs of light chains in a 1:1:1 stoichiometry. Myosin purified from amoebae grown in medium containing [32P]phosphate had two of the subunits labeled (0.2-0.3 mol of phosphate per mol of 210,000-dalton heavy chains and approximately 0.1 mol of phosphate per mol of 18,000-dalton light chain). Kinase activities specific for the 210,000-dalton and for the 18,000-dalton subunits have been identified in extracts of Dictyostelium amoebae, and the heavy chain kinase has been purified 50-fold. This kinase phosphorylated Dictyostelium myosin to a maximum of 0.5-1.0 mol of phosphate per mol of heavy chain. Heavy chain phosphate, but not light chain phosphate, can be removed with bacterial alkaline phosphatase. Actin-activated myosin ATPase increased 80% when phosphorylated myosin was dephosphorylated to a level of approximately 0.06 mol of phosphate per mol of heavy chain. This effect could be reversed by rephosphorylating the myosin. The ability of myosin to self-assemble into thick filaments was inhibited by heavy chain phosphorylation. For example, in 80-100 mM KCl, only 10-20% of the myosin was assembled into thick filaments when the heavy chains were fully phosphorylated. Removal of the heavy chain phosphate resulted in 70-90% thick filament formation. This effect on self-assembly could be reversed by rephosphorylating the dephosphorylated myosin. These findings suggest that heavy chain phosphorylation may regulate cell contractile events by altering the state of myosin assembly.
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PMID:Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments. 645 32

The major plasma membrane proteins of rabbit neutrophils were characterized by SDS-PAGE, surface iodination, 125I-concanavalin A binding, and detergent extraction. Neutrophil membranes were prepared which lacked significant intracellular contamination with good retention of protease-sensitive proteins. The major protein and predominant Con A-binding protein was as surface exposed, 140,000 D (gp 140) protein which was solubilized by nonionic detergents but not low ionic strength. Actin and myosin but not other cytosol proteins were prominently associated with the isolated membrane particularly in a Triton-insoluble form. Membranes were also prepared from surface-iodinated neutrophils previously stimulated with a chemotactic peptide or degranulated. The granule membrane enzyme alkaline phosphatase was incorporated into the plasma membrane fraction of degranulated neutrophils. However, the membrane proteins in the different membrane preparations were identical on SDS-PAGE and autoradiography. Therefore, using these techniques, no major alterations in protein composition of the plasma membrane could be detected following stimulation or degranulation of rabbit neutrophils.
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PMID:Characterization of rabbit neutrophil membrane proteins. A 140K major membrane protein is the predominant Con A-binding protein. 658 13


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