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W Y Cheung

Publications and source records attributed to W Y Cheung.

At least 55 records · Page 3Linked to original sources
PubMed1988

Demonstration of endogenous inhibitors for the formation of 14,15-oxido-5,8,11-eicosatrienoic acid from phosphatidylinositol in bovine lung extract.

Bovine lung extract contained an enzyme or enzyme system that catalyzed the formation of 14,15-oxido-5,8,11-eicosatrienoic acid from phosphatidylinositol. The enzyme activity increased markedly during the course of purification, apparently due to the removal of two endogenous inhibitors from the enzyme. Using membranes with known molecular weight cut-offs, we estimated the Mr of Inhibitor 1 to be between 10,000 and 100,000 and Inhibitor 2 less than 1,000. Inhibitor 1 appeared to be partially inactivated by trypsin and was heat labile, whereas Inhibitor 2 was resistant to trypsin and was heat resistant. Both inhibitors were hydrophilic.

8,11,14-Eicosatrienoic Acid↗
PubMed1988

The structure and regulation of phosphoglucose isomerase in Saccharomyces cerevisiae.

We have cloned and sequenced the PGI1 gene, encoding phosphoglucose isomerase (E.C.5.3.1.9), from Saccharomyces cerevisiae. The nucleotide sequence predicts subunits of 554 amino acids with a molecular weight of 61,230. Both the size and amino acid composition correlate well with measurements from purified protein. We have compared the PGI1 protein with the predicted sequence for pig muscle PGI. In spite of some evolutionary divergence the proteins are very similar and there are some highly conserved regions, two of which have been implicated in the active site. It has been suggested that PGI exists in two or more isozyme forms in S. cerevisiae and analogy with ADR2/ADC1 suggests that such PGI isozymes might also be differentially regulated during glycolytic/gluconeogenic growth. We have used accurate quantitation of PGI1 mRNA and gene fusions of PGI1 to the lacZ gene of Escherichia coli to show that PGI1 transcription is regulated neither between glycolytic and gluconeogenic growth nor between exponential and stationary phase. The complete lack of PGI activity in PGI1 deletion mutants and of differential regulation suggests that the isozymes of PGI might result merely from processing of the PGI1 gene product.

Amino Acid Sequence↗
PubMed1988

Quantitative subcellular localization of calmodulin-dependent phosphatase in chick forebrain.

Using a radioimmunoassay, we have measured the level of calmodulin-dependent phosphatase (calcineurin) in various subcellular fractions from chick forebrain. Our results revealed high levels of the enzyme in the cytoplasm and microsomes. A considerable amount was also observed in synaptosomes, where it was found exclusively in the synaptoplasm, comprising 0.32% of the total synaptoplasmic protein. Immunocytochemical localization of the phosphatase in isolated synaptosomes supported the biochemical finding. Phosphatase was not detected in nuclei, myelin, synaptic vesicles, and mitochondria. These results suggest that myelin basic protein and histone H1, widely used in biochemical characterization studies of the phosphatase, may not be physiological substrates, and that the cytoplasm, microsomes, and synaptoplasm may prove to be useful sources for the identification of physiological substrates.

Animals↗
PubMed1987

Differential reactivities of lysines in calmodulin complexed to phosphatase.

Calmodulin and calmodulin complexed with calcineurin phosphatase were trace labeled with [3H]acetic anhydride and the incorporation of [3H]acetate into each epsilon-amino lysine of calmodulin was measured. The relative reactivities of calmodulin lysines were higher in the presence of Ca2+ than in the presence of EGTA, and the order was: Lys-75 greater than Lys-94 greater than Lys-148 greater than or equal to Lys-77 greater than Lys-13 greater than or equal to Lys-21 greater than Lys-30. The changes in relative reactivity implied a change in conformation. When calmodulin was complexed with the phosphatase, Lys-21, Lys-77, and Lys-148 were most protected, implying that these residues are at or near the interaction sites or are conformationally perturbed by the interaction. Lys-30 and Lys-75 were slightly protected, lysine 13 showed no change, while lysine 94 significantly increased in reactivity. Comparison with results obtained from myosin light chain kinase using a similar technique (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232) reveals that calmodulin may interact with each of the two enzymes similarly at or near Lys-21, Lys-75, and Lys-148; one difference with phosphatase is that complex formation also involved Lys-77. These findings suggest that calmodulin interacts differently with its target enzymes.

Acetates↗
PubMed1987

Effects of cadmium on human platelet reactions.

Human platelets incubated with Cd2+ took up the cation slowly, and the uptake was speeded up by ionophore A23187. The capacity of human platelets to accumulate Cd2+ was large, equivalent to 10 nmol Cd2+ per mg protein. The effects of Cd2+ on protein phosphorylation and serotonin release of human platelets were studied. Washed platelets incubated with Cd2+ showed a general increase in protein phosphorylation concurrent with a slow release of serotonin. In the presence of ionophore A23187, however, Cd2+ had a biphasic effect on protein phosphorylation: stimulatory at low and inhibitory at high Cd2+ concentrations. The phosphorylation of two proteins with molecular masses close to 43 and 20 kDa was more sensitive to the inhibitory effect of Cd2+, and under similar conditions, the primary effect of Cd2+ on serotonin release was inhibitory, although at lower Cd2+ concentrations a slight stimulation was noted. Thrombin increased the phosphorylation of several proteins, and a prior incubation with Cd2+ further augmented that of a 20 kDa protein, but this treatment did not affect thrombin-induced serotonin release.

Blood Platelets↗
PubMed1987

Formation of cis-14,15-oxido-5,8,11-icosatrienoic acid from phosphatidylinositol in human platelets.

Human platelets contain a soluble enzyme or enzyme system that catalyzes the formation of lysophosphatidylinositol and a compound more polar than arachidonic acid (compound A) from 2-arachidonoyl sn-phosphatidylinositol. Arachidonic acid, 2-arachidonoyl sn-phosphatidylcholine, or 2-arachidonoyl sn-phosphatidylethanolamine did not serve as substrate for the production of compound A. The reaction required Ca2+ and was not affected by aspirin, indomethacin, or mepacrin. Enzyme activity was not enhanced in the presence of NADPH, but it was inhibited greater than 90% by CO or N2; inhibition was readily reversible by exposure to atmospheric air. Neither metapyrone (SKF 525A) nor cyanide, inhibitors of cytochrome P-450, inhibited compound A formation, suggesting that a cytochrome P-450 system was not involved. Thrombin stimulated the formation of compound A in whole platelets; ionophore A23187 did so much less effectively; and other agonists such as collagen, ADP, and epinephrine were ineffective. Compound A exhibited a fragmentation pattern by GC/MS identical to that of authentic cis-14,15-oxido-5,8,11-icosatrienoic acid. Collectively, these data indicate that human platelets may contain an enzyme system that catalyzes the epoxidation of the arachidonic acid moiety of phosphatidylinositol and its hydrolysis to liberate cis-14,15-oxido-5,8,11-icosatrienoic acid.

8,11,14-Eicosatrienoic Acid↗
PubMed1986

Substrate-specific forms of human platelet phospholipase A2.

Purification of human platelet phospholipase A2 (PLA2) from a particulate fraction by ion-exchange chromatography at 4 degrees C yielded a single peak of enzyme activity, which catalyzed the hydrolysis of arachidonic acid from the 2-position of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). The activity toward PtdCho and that toward PtdEtn differed in stability during storage, pH optimum, Ca2+ requirement, and affinity for the substrate; however, each activity preferred phospholipid with arachidonate at the 2-position. The two activities appeared to be eluted as an aggregate in a single peak from the ion-exchange column. When the column was run at 22 degrees C, an additional PLA2 activity peak specific for PtdEtn was resolved from the original PLA2 peak. But when the particulate fraction was briefly sonicated in 0.1% octylglucoside before chromatography at 22 degrees C, a different PLA2 activity peak, specific for PtdCho, was obtained. Resolution of the two specific forms of PLA2 under different conditions probably resulted from selective solubilization of the aggregate. The specific PLA2 activities thus isolated were very labile, whereas those in the aggregate were relatively stable. These findings suggest that human platelets contain at least two substrate-specific forms of PLA2, one for PtdCho and another for PtdEtn.

Blood Platelets↗
PubMed1985

Bovine brain calmodulin-dependent protein phosphatase. Regulation of subunit A activity by calmodulin and subunit B.

Calmodulin-dependent protein phosphatase isolated from bovine brain consists of a catalytic subunit A (Mr = 60,000) and a regulatory subunit B (Mr = 19,000) present in equal molar ratios. The two subunits were dissociated by gel filtration in 6 M urea and reconstituted to investigate the role of calmodulin and subunit B in regulating the phosphatase activity of subunit A. The activity of subunit A was stimulated 2-fold by calmodulin, 13-fold by subunit B, and 21-fold by both, indicating that the effects of both were synergistic. Maximum stimulation by calmodulin was observed at a calmodulin to subunit A molar ratio of 2:1 in the presence or absence of subunit B, whereas that by subunit B was observed at a B to A molar ratio of 3:1 in the presence or absence of calmodulin. Calmodulin and subunit B increased the Vmax of subunit A 2- and 5-fold, respectively, but had little effect on the Km for casein. The specific activity of the phosphatase reconstituted from subunits A and B reached 86% that of the native enzyme, whereas that of the holoenzyme reached 90%. Subunit B, even though similar to calmodulin in many respects, did not stimulate the activity of native phosphatase, suggesting that it cannot substitute for calmodulin. Limited trypsinization of subunit A increased its catalytic activity to the level observed with calmodulin; and this activity was further stimulated by subunit B but not by calmodulin. These results indicate that subunit A of phosphatase contains one catalytic domain and two distinct regulatory domains, one for calmodulin, and another for subunit B, that these two proteins do not substitute for one another and that they stimulate subunit A synergistically.

Animals↗
PubMed1985

Stimulation of calmodulin by cadmium ion.

Cd2+, a serious environmental pollutant in certain industrial regions, accumulates in mammalian tissues with a very slow turnover. Using various criteria, we studied the ability of Cd2+ to substitute for Ca2+ in calmodulin (CaM), a ubiquitous Ca2+-binding protein that mediates many of the Ca2+ effects. CaM bound Cd2+ with a Kd of 4.5 microM, presumably to the Ca2+-binding sites. Binding of Cd2+ allowed CaM to bind 2 moles chlorpromazine, or to form a complex with skeletal muscle troponin-I, troponin-T, or phosphodiesterase. Complex formation with phosphodiesterase led to its activation, which was observed even in the presence of glutathione or cysteine, agents known to chelate Cd2+. This raises the possibility that one manifestation of Cd2+ toxicity may be through its activation of CaM, thus upsetting its normal regulation by a cellular flux of Ca2+.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Inhibition of human platelet phospholipase A2 activity by unsaturated fatty acids.

Phospholipase A2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) activity from human platelets increases significantly when the enzyme is separated from an endogenous inhibitor(s). The inhibitor, associated mainly with a particulate fraction, has now been identified as a mixture of unsaturated fatty acids. Treatment of the inhibitor with trypsin, RNase, DNase, or heat did not diminish its inhibitory activity, which was extractable by organic solvents. Incubation of PLA2 with phospholipids or various neutral lipids, including saturated fatty acids, had little or no effect on enzymatic activity. In contrast, unsaturated fatty acids such as palmitoleic acid (16:1), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), all of which were detected in the particulate fraction, or longer chained unsaturated fatty acids inhibited PLA2 activity by 50% at approximately equal to 5 X 10(-7) M. The level of unsaturated fatty acids in the inhibitor fraction was equivalent to approximately equal to 10(-4) M, apparently sufficient to effectively inhibit PLA2 activity. Methylation of unsaturated fatty acids caused a complete loss of inhibitory activity, and subsequent demethylation restored the activity, suggesting that a free carboxyl group was necessary. Inhibition of PLA2 by unsaturated fatty acids appeared to be noncompetitive. PLA2 absolutely required Ca2+ for activity; the inhibition by unsaturated fatty acids was not reversed by Ca2+. The finding that unsaturated fatty acids are potent inhibitors of PLA2 would explain its generally low activity in human platelet extracts and its marked increase of activity during the course of enzyme purification.

Blood Platelets↗
PubMed1985

Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina.

Calmodulin-dependent protein phosphatase, previously called CaM-BP80 or calcineurin, is present in high concentrations in the central nervous system. The level of the phosphatase has been shown by radioimmunoassay to increase during development in the retinas of embryonic and hatching chicks (Tallant, E.A., and W.Y. Cheung, 1983, Biochemistry, 22:3630-3635). The aims of this study are to immunocytochemically localize the phosphatase in developing and mature retinas and to determine if the phosphatase is present in fractions of retinal synaptic membranes and synaptic junctions. Vibratome slices of fixed chick retina and Western blots of detergent-solubilized retinal fractions are both treated sequentially with rabbit primary antisera and goat anti-rabbit Fab fragments conjugated to peroxidase, and then reacted with hydrogen peroxide and diaminobenzidine. The tissue slices are further processed for electron microscopy. This paper demonstrates the presence of peroxidase reaction product in the retina just before synapse formation. In the outer plexiform layer the product is confined to photoreceptor synaptic terminals, whereas in the inner plexiform layer it is present in synaptic terminals of bipolar cells and in dendrites of ganglion cells. In this latter site the product is present postsynaptically at bipolar and amacrine synapses. The phosphatase is detected in Western blots of both synaptic plasma membrane and synaptic junction fractions.

Animals↗
PubMed1984

Subunit A of calmodulin-dependent protein phosphatase requires Mn2+ for activity.

Bovine brain calmodulin-dependent protein phosphatase comprises a catalytic subunit A (Mr 60,000) and a regulatory subunit B (Mr 19,000). The native enzyme was active with Ca2+ or Mn2+. Upon resolution into its subunits in 6 M urea and 15 mM EDTA, subunit A was active with Mn2+; Co2+ and Ni2+ partially substituted for Mn2+, but Ca2+, Mg2+ and Zn2+ were ineffective. The stimulating effect of Mn2+ was not easily reversed by EGTA. Like the native phosphatase, subunit A was markedly stimulated by calmodulin or by controlled trypsinization. Unlike the native enzyme, however, trypsinized subunit A still required Mn2+ for activity. These findings provide evidence that the catalytic subunit of phosphatase may be a metallo (possibly Mn2+) enzyme.

Animals↗
PubMed1984

Activation of bovine brain calmodulin-dependent protein phosphatase by limited trypsinization.

A calmodulin-dependent protein phosphatase isolated from bovine brain [Tallant, E.A., & Cheung, W.Y. (1983) Biochemistry 22, 3630-3635] is stimulated by limited trypsinization to the same activity level as that by calmodulin. Prolonged trypsinization caused gradual loss of phosphatase activity, a process retarded in the presence of Ca2+, and even more in the presence of calmodulin. Trypsinized phosphatase, when fully activated, had a molecular weight of 60 000 and was composed of two protein species of 43 000 and 16 000 daltons. Trypsinization decreased the Km of phosphatase for casein from 10.8 to 1.2 microM and increased the Vmax from 4.9 to 30.9 nmol (mg of protein)-1 min-1. The proteolyzed enzyme was insensitive to calmodulin and did not bind to a calmodulin-Sepharose affinity column. It was, however, stimulated by Ca2+, requiring 0.4 microM Ca2+ for half-maximal activation. Both native and trypsinized phosphatase were stimulated by Mn2+ to a level considerably higher than that by Ca2+.

Animals↗