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W Y Cheung

Publications and source records attributed to W Y Cheung.

At least 73 records · Page 4Linked to original sources
PubMed1984

Characterization of bovine brain calmodulin-dependent protein phosphatase.

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.

Animals↗
PubMed1984

Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A.

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.

Animals↗
PubMed1984

Activation of calmodulin by various metal cations as a function of ionic radius.

The active form of calmodulin is a Ca2+ . calmodulin complex. The purpose of this investigation was to determine whether other metal cations substitute for Ca2+ to activate calmodulin. Binding of Ca2+ resulted in an altered conformation of calmodulin with an increased quantum yield in its tyrosine fluorescence. Qualitatively similar results were obtained with Zn2+, Mn2+, Cd2+, Hg2+, Sr2+, Pb2+, Tb3+, Sm3+, and La3+. The relative extents of fluorescence enhancement by these cations were related to their ionic radii: all cations with ionic radii close to Ca2+ (0.99 A) increased tyrosine fluorescence, whereas those with different ionic radii were not effective, or much less so. The change in calmodulin conformation by the cations was confirmed by its altered electrophoretic mobility on polyacrylamide gels. Cations that change the conformation of calmodulin allow it to stimulate phosphodiesterase. The relative extents of stimulation of phosphodiesterase by cations were also related to their ionic radii. Finally, the ability of metal cations to inhibit Ca2+ binding was similarly related to their ionic radii. In general, the closer the radius of a metal cation was to that of Ca2+, the more effective was the cation to substitute for Ca2+. The range of effective ionic radii was approximately 1 +/- 0.2 A. Calmodulin-stimulated phosphodiesterase activity by the cations was reversed by trifluoperazine, an antagonist of calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Calmodulin: its potential role in cell proliferation and heavy metal toxicity.

Increasing evidence indicates that calmodulin, the ubiquitous Ca2+-binding protein in eukaryotes, serves as a major intracellular Ca2+ receptor regulating the activity or rate of many key enzymes and cellular processes, including a potential role in cell proliferation. Certain heavy metals are highly toxic to the cell; preliminary findings from our laboratory suggest that these metals effectively substitute for Ca2+ in calmodulin. Activation of calmodulin by these metals could upset its normal regulation by the cellular flux of Ca2+. Perhaps this could constitute in part a basis for the toxicity of these metals in cell physiology.

Animals↗
PubMed1983

Calmodulin-dependent protein phosphatase: a developmental study.

Calmodulin-dependent protein phosphatase, one of the major calmodulin-binding proteins in bovine brain, dephosphorylates casein with a specific activity of 15 nmol mg-1 min-1 at 30 degrees C. The stimulation of phosphatase activity by calmodulin is reversed by ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or trifluoperazine, a calmodulin antagonist. Antibodies raised in rabbit against the phosphatase inhibit the enzyme activity. The levels of the protein in brain extracts from various animals, determined by a radioimmunoassay, range from 20 micrograms/g of tissue in chick and fish brains to 143 micrograms in rat cerebrum. The ontogeny of the phosphatase was studied in nervous tissues from rat and chick, animals in which synaptogenesis takes place at different times during their development. The levels of the protein increased significantly in rat cerebrum and cerebellum and in chick brain and retina during the periods corresponding to major synapse formation. In rat cerebrum, the enzyme appeared to be equally distributed between the cytosol and the particulate fraction; the level in both compartments increased during the major period of synapse formation. Thus, the development of calmodulin-dependent protein phosphatase closely parallels synaptogenesis, implicating a role in some synaptic function.

Aging↗
PubMed1983

Immunofluorescent localization of cGMP, cGMP-dependent protein kinase, calmodulin and cAMP in the rat uterus. Effects of estrogen treatment.

Cyclic guanosine 3',5' monophosphate (cGMP), cGMP-dependent protein kinase, calmodulin and cyclic adenosine 3',5' monophosphate (cAMP) were localized in the uterus of the immature rat by an indirect immunofluorescence technique. cGMP, cGMP-dependent protein kinase and calmodulin were detected predominantly along epithelial and myometrial plasma membranes and in the adjacent cytoplasm. In contrast, cAMP immunoreactive material was found principally in the cytoplasm of connective tissue. After administration of 17 beta-estradiol, similar time-dependent changes were observed in the localization of cGMP, cGMP-dependent protein kinase and calmodulin in all uterine cell types. For the three compounds, nucleolar-like distribution of the immunofluorescence appeared approximately 12 h after treatment. A more dispersed, reticular distribution of the nuclear fluorescent staining was observed 20-24 h after hormonal treatment. Estrogen did not affect the localization of cAMP. The simultaneous mobilization of cGMP, cGMP-dependent protein kinase and calmodulin towards the same nuclear loci suggests concerted roles for these three molecules in nuclear metabolic processes during the development of the uterotrophic action of estrogens.

Animals↗
PubMed1983

Marked increase of human platelet phospholipase A2 activity in vitro and demonstration of an endogenous inhibitor.

When soluble and particulate fractions of human platelet homogenate were chromatographed on DEAE-cellulose and hydroxylapatite columns, total phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity increased sharply. PLA2 activity detected in these partially purified preparations was approximately 12 times greater than that associated with the original homogenate. Chromatography of the particulate enzyme on a DEAE-cellulose column yielded one activity peak. Fractions eluted near the activity peak showed a trace of PLA2 activity but inhibited purified PLA2 stoichiometrically, suggesting the presence of an endogenous "inhibitor" in the homogenate. The inhibitor activity was heat-stable, trypsin insensitive, and extractable by chloroform/methanol, and thus appears to be associated with a lipid(s). PLA2 activity was Ca2+ dependent. The crude enzyme was variably stimulated by calmodulin, whereas the purified enzyme was not, suggesting that the effect of calmodulin on the crude enzyme is indirect. Our results suggest that human platelet PLA2 activity reported in the literature may have been underestimated, apparently due to the presence of an endogenous inhibitor.

Blood Platelets↗
PubMed1982

Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence.

Terbium, a trivalent lanthanide, effectively substituted for Ca2+ in calmodulin as judged by several criteria: intrinsic fluorescence spectra, altered mobilities on polyacrylamide gel electrophoresis, formation of a stable complex with troponin I or calcineurin, and stimulation of phosphodiesterase. Calmodulin harbors four Ca2+ binding domains; domains I and II contain no tyrosine, whereas domains III and IV each have one tyrosine. The binding of Tb3+ to calmodulin was followed by the increase of Tb3+ fluorescence at 545 nm upon binding to calmodulin. This fluorescence was elicited either by exciting Tb3+ directly at 222 nm or by exciting the calmodulin tyrosine at 280 nm with resulting energy transfer from tyrosine to Tb3+. Fluorescence generated by direct excitation measures binding of Tb3+ to any of the Ca2+ binding domains, whereas energy transfer through indirect excitation is effective only when Tb3+ is within 5 A of tyrosine, indicating that Tb3+ necessarily occupies a Ca2+ binding domain that contains tyrosine. A judicious use of the direct and indirect excitation could reveal the sequence of fill of the binding domains. Our results suggest these domains are filled in the following sequence: 1) domain I or II; 2) domains III and IV; and 3) domain II or I that has not been filled initially.

Animals↗
PubMed1982

Calmodulin.

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Adrenal Cortex↗
PubMed1982

Calmodulin: an overview.

Calmodulin is a 16,700-dalton Ca2+-binding protein ubiquitous in the eukaryotes. It has no intrinsic enzymatic activity, but it regulates a wide spectrum of enzymes that control many basic cellular processes, ranging from the metabolism of cyclic nucleotides, Ca2+, and glycogen to contractile activity and stimulus-secretion coupling. Mounting evidence now indicates that calmodulin is the major intracellular Ca2+ receptor that remained elusive despite three decades of extensive work by many investigators.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗