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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.
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PMID:Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins. 629 46

The actin-activated ATPase activity of myosin II from Acanthamoeba castellanii is inhibited by phosphorylation of 3 serine residues near the carboxyl end of the heavy chain of the molecule. We have purified a protein phosphatase from Acanthamoeba using myosin II as a substrate. This phosphatase has a molecular weight of 39,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point in urea of 5.2. The enzyme also is active against other phosphoserine protein substrates such as turkey gizzard smooth muscle myosin light chain, but not against a synthetic phosphotyrosine protein substrate. It does not hydrolyze ATP or p-nitrophenol phosphate. No effector has been found to increase substantially the activity of the enzyme as isolated, but it is inhibited by ATP, pyrophosphate, and NaF. This inhibition is reduced in the presence of MnCl2. The Mg2+-dependent actin-activated ATPase of myosin II is activated by dephosphorylation of phosphorylated myosin II by the phosphatase. Its broad substrate specificity, molecular weight, and response to protein phosphatase inhibitors suggest that the Acanthamoeba protein phosphatase is a type 2A phosphatase (Cohen, P. (1982) Nature (Lond.) 206, 613-620).
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PMID:Purification of a protein phosphatase from Acanthamoeba that dephosphorylates and activates myosin II. 631 29

The calmodulin-dependent protein phosphatase from bovine brain is composed of two subunits: subunit A, Mr 60,000, and subunit B, Mr 16,500. Using in vitro immunization techniques, we have produced a monoclonal antibody specific for the phosphatase. The antibody was immobilized to Sepharose 4B to prepare an immunoabsorbent column, which was used to purify the enzyme. Phosphatase isolated from the column showed a polypeptide with Mr 60,000, equivalent to subunit A, which showed calmodulin-dependent phosphatase activity. Subunit B was not obtained from the column. Limited trypsin digestion stimulated phosphatase activity, yielding polypeptides of Mr 59,000, 43,000, and 16,000; the phosphatase activity after trypsin digestion was calmodulin independent. Chromatography of trypsin-treated phosphatase on an immunoaffinity column yielded two proteins, Mr 59,000 and 43,000, that were catalytically active and calmodulin independent. In a separate experiment, the two subunits of the phosphatase were separated by gel filtration in 6 M urea. Subunit A isolated from the filtration column showed little or no activity in the presence of Ca2+ and calmodulin, but it showed calmodulin-dependent phosphatase activity in the presence of 0.8 mM Mn2+. Subunit B was catalytically inactive. Collectively, these results indicate that subunit A and its proteolytic fragment contain the catalytic site and the antigenic determinant for the monoclonal antibody.
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PMID:Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A. 632 93

Phosphorylation of thylakoid membrane proteins in the chloroplast of wild-type and mutant strains of Chlamydomonas reinhardi has been studied in vivo and in vitro. Intact cells or purified membranes were labeled with [32P]orthophosphate or [gamma-32P]ATP, respectively, and the presence of phosphorylated polypeptides was detected by autoradiography after membrane fractionation by SDS PAGE. The 32P was esterified to serine and threonine residues. At least six polypeptides were phosphorylated in vitro and in vivo, and corresponded to components of the photosystem II complex contributing to the formation of the light-harvesting-chlorophyll (LHC) a,b-protein complex, the DCMU binding site (32-35 kdaltons), and the reaction center (26 kdaltons). In agreement with previous reports (Alfonzo, et al., 1979, Plant Physiol., 65:730-734; and Bennett, 1979, FEBS (Fed. Eur. Biochem. Soc.) Lett., 103:342-344), the membrane-bound protein kinase was markedly stimulated by light in vitro via a mechanism requiring photosystem II activity. Phosphorylation of thylakoid membrane polypeptides in vivo was, however, completely independent of illumination. Similar amounts of phosphate were incorporated into the photosynthetic membranes of cells incubated in the dark, in white light with or without 3-(3,4-dichlorophenyl-1,1-dimethyl urea (DCMU), or in red or far-red light. Different turnovers of the phosphate were observed in the light and dark, and a phosphoprotein phosphatase involved in this turnover process was also associated with the membrane. Comparison of the amount of esterified phosphate per protein in vivo and the maximum incorporation in isolated membranes revealed that only a small fraction of the available sites could be phosphorylated in vitro. In contrast to the DCMU binding site, the LHC and 26-kdalton polypeptide were not phosphorylated in vivo when the reaction center II polypeptides of 44-54 kdaltons were missing. The finding that all the phosphoproteins appear to be components of the photosystem II complex and are only partially dephosphorylated in vivo suggests strongly that protein phosphorylation might play an important role in the maintenance of the organizational integrity of this complex. The observation that the LHC is not phosphorylated in the absence of the reaction center lends support to this idea.
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PMID:Phosphorylation of chlamydomonas reinhardi chloroplast membrane proteins in vivo and in vitro. 681 97

We examined the effect of urea on NaK2Cl cotransport in human erythrocytes. In erythrocytes from nine normal subjects, the addition of 45 mM urea, a concentration commonly encountered in uremic subjects, inhibited NaK2Cl cotransport by 33 +/- 7%. Urea inhibited NaK2Cl cotransport reversibly, and in a concentration-dependent fashion with half-maximal inhibition at 63 +/- 10 mM. Acute cell shrinkage increased, and acute cell swelling decreased NaK2Cl cotransport in human erythrocytes. Okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, increased NaK2Cl cotransport by nearly 80%, suggesting an important role for these phosphatases in the regulation of NaK2Cl cotransport. Urea inhibited bumetanide-sensitive K influx even when protein phosphatases were inhibited with OA, suggesting that urea acted by inhibiting a kinase. In cells subjected to shrinking and OA pretreatment, maneuvers expected to increase the net phosphorylation, urea inhibited cotransport only minimally, suggesting that urea acted by causing a net dephosphorylation of the cotransport protein, or some key regulatory protein. The finding that concentrations of urea found in uremic subjects inhibited NaK2Cl cotransport, a widespread transport pathway with important physiological functions, suggests that urea is not only a marker for accumulation of other uremic toxins, but may be a significant uremic toxin itself.
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PMID:Urea inhibits NaK2Cl cotransport in human erythrocytes. 759 97

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin28kDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33% of total (SDS-soluble) calbindin28kDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskeletal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDa and calcineurin demonstrated here provides the first molecular evidence that enamal cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.
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PMID:Calbindin28kDa and calmodulin are hyperabundant in rat dental enamel cells. Identification of the protein phosphatase calcineurin as a principal calmodulin target and of a secretion-related role for calbindin28kDa. 760 Nov 26

Organ transplantation offers new life to patients who suffer from incurable disease. The problem of rejection of the transplanted organ has been overcome with the use of potent immunosuppressive drugs. These drugs, although they allow graft tolerance and graft survival, also are associated with complications such as osteoporosis. Although factors such as nutrition, gonadal status, and ambulatory status are important, the use of immunosuppressive drugs appears to be the main factor in the development of osteoporosis. The drugs that are responsible for this bone loss are glucocorticoids and the calcineurin phosphatase inhibitors, cyclosporine and tacrolimus. The incidence of bone disease depends, in part, on which organ is transplanted. Kidney transplant recipients appear to be less susceptible to the development of overt osteoporosis than do heart or liver transplant recipients. The most critical period of bone loss in organ recipients appears to be within the first 6 months, with the most dramatic reduction occurring within the first 3 months following transplantation. Trabecular (cancellous) bone of the spine appears to be most at risk, with vertebral fractures occurring most commonly. Transplant recipients should be evaluated by bone mineral densitometry and measurement of vitamin D metabolites, blood urea nitrogen, creatinine, calcium, and phosphate. Markers of bone turnover may help in assessing the rate of remodeling. Gonadal function should be ascertained by measurement of serum testosterone (males) or estradiol (females) levels. Therapy should be directed toward prevention of bone loss as well as helping to restore what already may have been lost. Administration of calcium and vitamin D and sex hormone replacement, if indicated, should be considered.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Organ transplantation and osteoporosis. 761 20

Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
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PMID:Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A. 855 May 70

K influx and efflux (both ouabain- and bumetanide-resistant) in haemoglobin S-containing red cells (sickle cells) were markedly stimulated by urea (> 0.25 M). Stimulation was rapid and reversible. Volume-sensitive KCl cotransport in both HbA or HbS red cells is thought to be O2-dependent but we show here that urea-stimulated K fluxes in sickle cells were largely insensitive to O2 tension. Urea-stimulated K fluxes were not inhibited by lowering the external Ca concentration (with EGTA) but were abolished by Cl-substitution (with MeSO4 or NO3) or pretreatment of cells with the protein phosphatase inhibitor, calyculin A (0.1 muM). Results are consistent with a stimulatory action of urea on the KCl cotransporter, independent of oxygen tension, mediated via the phosphorylation cascade which regulates the transporter. The importance of this effect to the physiology and pathology of sickle cells is discussed.
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PMID:Effects of urea and oxygen tension on K flux in sickle cells. 947 28

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.
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PMID:Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors. 1079 19


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