Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P67775 (alpha isoform)
 797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The alpha isoform of phosphatidylinositol-specific phospholipase C (alpha-PI-PLC, Mr 62,000) was purified from bovine brain. Enzyme activity was dependent on calcium, sodium cholate and showed the anticipated specificity for the phosphatidylinositols. Calcium interaction with this protein, investigated by gel filtration chromatography, showed no detectable binding at calcium concentrations adequate to activate the enzyme. Association of alpha-PI-PLC with phospholipid vesicles was studied by light scattering, fluorescence energy transfer and gel-filtration chromatography. The enzyme readily associated with vesicles of high charge density, with vesicles of crude acidic phospholipids and with PIP2. Interaction was characterized by a rapid association followed by slower addition of more protein to the phospholipid. Complexes containing 20-30 percent protein (by weight) were readily obtained. Calcium had only a small effect on this interaction. The protein-phospholipid complexes appeared to bind less calcium than a similar amount of phospholipid alone. Thus, alpha-PI-PLC did not appear to be a calcium-binding protein in either its free or membrane-associated states. Although alpha-PI-PLC showed the highest propensity to bind to phospholipids, a number of other proteins also associated with phospholipids under the conditions used. Thus, whether or not the observed interaction of alpha-PI-PLC with membranes was specific and biologically important or whether it was a process common to many proteins, was not known. Knowledge of this interaction may enhance our understanding of possible mechanisms for protein-membrane interactions in general.
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PMID:Association of alpha-phosphatidylinositol-specific phospholipase C with phospholipid vesicles. 131

The carboxyl-terminal isoforms of troponin T (TnT), alpha and beta, differing in the sequence of a region near the COOH terminus, arise from alternative splicing of a primary transcript of the TnT gene and are expressed in a tissue-specific and developmentally regulated manner (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). To date, the beta isoform has not been studied directly at the protein level. To explore the potential functional differences between the alpha and beta sequences, we isolated two rabbit skeletal TnT cDNA clones: a full-length cDNA for a beta isoform and a partial-length cDNA for an alpha isoform. Two restriction fragments derived from the cDNA clones were used to direct overexpression, in Escherichia coli, of two TnT fragments, T2p-alpha and T2p-beta, each containing the last 108 amino acid residues of the alpha or beta isoform of TnT. Using purified T2p-alpha and T2p-beta along with fluorescent derivatives of troponin C (TnC) and alpha alpha-tropomyosin (Tm), we showed that T2p-alpha bound more strongly to TnC than did T2p-beta both in the presence and absence of Ca2+, and exhibited a higher affinity for Tm than did T2p-beta. More interestingly, the Ca2+ affinities of the Ca(2+)-specific regulatory sites of TnC in the T2p-alpha. TnC complex were found to be 3-fold higher than in T2p-beta.TnC complex. These results support the hypothesis that the sequence divergence between the alpha and beta isoforms of TnT may have functional significance in possibly contributing to the determination of the Ca2+ sensitivity of muscle fibers.
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PMID:Two genetically expressed troponin T fragments representing alpha and beta isoforms exhibit functional differences. 142 53

Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The beta isoform of PKC is translocated and degraded much more rapidly than the alpha isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC alpha and beta are strikingly different in antigen-activated RBL cells. PKC beta associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC alpha concentrates in regions of the cell periphery. This distribution of PKC beta is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC beta and to the intermediate filament protein vimentin are almost identical, indicating that PKC beta associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC beta with its substrates and thus confer specificity to the actions of this isoform.
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PMID:Association of the beta isoform of protein kinase C with vimentin filaments. 151 48

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC alpha isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000-39,000, the 24,000 and the triplet of 17,000-20,000-22,000 molecular weight. The addition of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 molecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes.
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PMID:Heterogeneity of protein kinase C in cultured rat mesangial cells. 161 24

The early events that occur after treatment of the highly interferon alpha (IFN-alpha)-sensitive human lymphoblastoid Daudi cell line with human leukocyte IFN-alpha have been examined. IFN-alpha treatment of Daudi cells results in a rapid and transient increase in the cellular content of diacylglycerol, which occurs in the absence of inositol phospholipid turnover, or an increase in intracellular calcium concentration. Furthermore, IFN-alpha treatment results in a selective, time-dependent activation of the Ca(2+)-independent epsilon isoform of protein kinase C (PKC), while the alpha isoform is unaffected by IFN-alpha treatment. In contrast, IFN-alpha treatment of an IFN-resistant subclone of Daudi cells had no effect on the diacylglycerol content of cells and on the activation of PKC-epsilon. The selective PKC inhibitor staurosporine blocked the transcriptional activation of IFN-alpha-stimulated genes, the cytoplasmic accumulation of mRNAs for these genes, and the induction of antiviral activity by IFN-alpha against vesicular stomatitis virus in IFN-sensitive cells. These observations suggest that transmembrane signaling of IFN-alpha involves diacylglycerol production and activation of PKC-epsilon in Daudi cells.
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PMID:Transmembrane signaling by interferon alpha involves diacylglycerol production and activation of the epsilon isoform of protein kinase C in Daudi cells. 183 72

Heat shock treatment of rat embryo fibroblasts resulted in a 60% increase in cytosolic protein kinase C activity, in contrast to phorbol ester-induced translocation to the membrane. During reversal of the cells back to the normal temperature a decrease in cytosolic PKC activity was observed and paralleled by an increase in protamine kinase activity. Cell lysates prepared from heat shock-treated cells show a marked calcium/phospholipid-dependent phosphorylation of several endogenous PKC substrate proteins, while the 28-kDa stress protein was shown to be a PKC substrate. These cells express the TYPE III-alpha isoform of PKC and, thus, the alterations induced within cells exposed to hyperthermic treatment may reflect a functional significance with regard to the regulation of this specific isoform.
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PMID:Alterations in protein kinase C type III-alpha during heat shock of rat embryo fibroblasts. 184 15

We have investigated the time course of expression of the alpha and beta triad junctional foot proteins in embryonic chick pectoral muscle. The level of [3H]ryanodine binding in muscle homogenates is low until day E20 of embryonic development, then increases dramatically at the time of hatching reaching adult levels by day N7 posthatch. The alpha and beta foot protein isoforms increase in abundance concomitantly with [3H]ryanodine binding. Using foot protein isoform-specific antibodies, the alpha foot protein is detected in a majority of fibers in day E10 muscle, while the beta isoform is first observed at low levels in a few fibers in day E15 muscle. A high molecular weight polypeptide, distinct from the alpha and beta proteins, is recognized by antifoot protein antibodies. This polypeptide is observed in day E8 muscle and declines in abundance with continued development. It appears to exist as a monomer and does not bind [3H]ryanodine. In contrast, the alpha isoform present in day E10 muscle and the beta isoform in day E20 muscle are oligomeric and bind [3H]ryanodine suggesting that they may exist as functional calcium channels in differentiating muscle. Comparison of the intracellular distributions of the alpha foot protein, f-actin, the heavy chain of myosin and titin in day E10 muscle indicates that the alpha foot protein is expressed during myofibril assembly and Z line formation. The differential expression of the foot protein isoforms in developing muscle, and their continued expression in mature muscle, is consistent with these proteins making different functional contributions. In addition, the expression of the alpha isoform during the time of organization of a differentiated muscle morphology suggests that foot proteins may participate in events involved in muscle differentiation.
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PMID:Foot protein isoforms are expressed at different times during embryonic chick skeletal muscle development. 202 50

A simple procedure is described for the purification of the alpha alpha isoform of S-100 proteins (S-100a0) from porcine heart. Purification steps include the following: i) extraction of the tissue with a hypotonic medium containing EDTA; ii) ammonium sulfate fractionation (0-50%) of the extract; iii) Ca2+-dependent affinity chromatography of the supernatant obtained through the preceding step on phenyl-sepharose and elution of absorbed proteins through a two-chamber gradient of 1.0-0.0 mM CaCl2 and 0.0--1.0 mM EGTA, respectively; and iv) chromatography of the resultant S-100-containing fractions on Sephadex G-200. The yield is 20 mg S-100a0/kg porcine heart. The whole procedure takes five days and is highly reproducible. Data obtained from the phenyl-sepharose step suggest that the affinity of Ca2+ for S-100a0 increases by several orders of magnitude once the protein had interacted with that matrix. This observation is discussed in relation to the role of S-100 proteins in amplification of the Ca2+ signal. Immunocytochemical and immunoblotting analyses indicate that S-100a0 is exclusively found at the level of the sarcolemmal membranes, the membranes of the sarcoplasmic reticulum, the external mitochondrial membranes, and in the adjacent sarcoplasm. No evidence of S-100a0 being associated with the nuclei or with myofibrils has been obtained. Finally, the cardiac tissue does not contain the Triton X-100-extractable fraction of S-100 normally detected in the brain and in adipocytes. Our data suggest that S-100a0 behaves as a peripheral membrane protein in cardiac tissue.
Cell Calcium
PMID:Cardiac S-100a0 protein: purification by a simple procedure and related immunocytochemical and immunochemical studies. 274 4

From the results of this study the following significant conclusions can be drawn. First, there are two separate inotropic responses to ouabain in the rat ventricle and the isolated rat heart resulting in a biphasic dose-response curve. Second, both the "low dose" and "high dose" inotropic responses of ouabain in the rat heart result from the inhibition of NKA, i.e., NKA is the binding site of ouabain for both "low" and "high" dose effects. Third, biphasic inotropic effects (rat ventricle) go together with alpha + and alpha (high and low affinity) isoforms of NKA, monophasic (rat atrium) with the alpha isoform (low affinity). Fourth, high extracellular calcium concentrations inhibit NKA and mask the full expression of the "low-dos" effect.
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PMID:The calcium dependence of the biphasic inotropic response to ouabain in the isolated rat heart. 283 73

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100-26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1-3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa) catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B alpha isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase alpha, and pyruvate kinase, and the Km values were 0.8 microM, 3.7 microM, and 2.2 microM, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K alpha value for Xu 5-P was 20 microM. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P2 level in liver after high glucose administration.
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PMID:Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. 759 45


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