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W Schook

Publications and source records attributed to W Schook.

At least 19 recordsLinked to original sources
PubMed1986

Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles.

Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).

Animals↗
PubMed1986

Cyclic nucleotide phosphodiesterase activity in bovine brain coated vesicles.

Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a Km of approximately 22 microM for cyclic AMP, a Vmax of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity. The enzyme activity was stimulated by cyclic GMP with maximal indexes of stimulation ranging between 40 and 300%. Both basal and stimulated phosphodiesterase activities were immunotitrated with polyclonal antibodies against clathrin attached to heat-inactivated, formaldehyde-fixed Staphylococcus aureus cells. The main form of phosphodiesterase activity present in the immunoprecipitated brain coated vesicle preparation also is stimulated by cyclic GMP. The allosteric behavior was modulated by cyclic GMP. All of these properties are typical of type II or cyclic GMP-sensitive phosphodiesterases in addition to their calcium and calmodulin independence. Competition experiments with a series of phosphodiesterase inhibitors, papaverine, 1-methyl-3-isobutylxanthine, and theophylline, showed inhibition of cyclic AMP hydrolysis. Trifluoperazine was inactive at the highest concentration used, 100 microM. These compounds also inhibited the cyclic GMP-stimulated cyclic AMP hydrolysis with trifluoperazine practically inactive. At 5 microM cyclic AMP none of the inhibitors was seen to stimulate the cyclic AMP hydrolytic activity. The presence of an enzyme for the breakdown of cyclic nucleotides in brain coated vesicles may suggest a role for these second messengers in the in vivo functions of this organelle.

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

Calcium-dependent binding of calmodulin to phospholipase A2 subunits induces enzymatic activation.

Calmodulin interacted with phospholipase A2 from two different sources, as established by affinity chromatography, dimethylsuberimidate protein crosslinking, and phospholipase A2 assays. Calmodulin was covalently crosslinked to pancreatic and bee venom phospholipases A2 in a calcium-dependent manner, and enhanced the enzymatic activities of these phospholipases. Pancreatic phospholipase A2 was separated into two species of identical molecular weight by calmodulin affinity chromatography; the species that bound to immobilized calmodulin in a calcium-dependent manner was stimulated by calmodulin. This presents further evidence that phospholipase A2 is directly activated by calmodulin.

Animals↗
PubMed1984

Regulation of endogenous calcium-dependent synaptic membrane phospholipase A2.

Synaptic plasma membrane preparations from brain tissue have endogenous Ca2+-dependent phospholipase A2 activity. Characterization of this activity revealed that it was maximally active at 10(-7)-10(-5) M Ca2+ and pH 7.0. The enzyme had a Km of 62.0 microM and a Vmax of 98.0 nmol/mg/h. Calmodulin and prostaglandin F2 alpha stimulated phospholipase A2 activity, whereas prostaglandin E2, cyclic AMP and ATP were inhibitory. Addition of exogenous phospholipase A2 to synaptic plasma membrane and synaptic vesicle preparations led to their disruption and/or lysis. We suggest that Ca2+-dependent regulation of phospholipase A2 activity may be required for synaptic vesicle and synaptic plasma membrane interaction.

Adenosine Triphosphate↗
PubMed1983

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides.

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.

Animals↗
PubMed1983

Preliminary characterization of synaptic vesicle/calmodulin interaction.

The association of calmodulin with brain synaptic vesicle proteins was analyzed. Scatchard analysis of [125I]calmodulin binding to brain synaptic vesicles revealed one high-affinity, low-binding-capacity, Kd = 1.0 (+/- 0.15) nM, Bmax = 4.1 (+/- 0.6) pmol/mg, and one low-affinity high-binding-capacity site, Kd = 177. (+/- 12.0) nM and Bmax = 202 (+/- 15.0) pmol/mg. Triton X-100 solubilization of synaptic vesicle proteins and subsequent elution on a Sepharose-4B-CNBr-calmodulin affinity column demonstrated that two protein doublets of approximate MrS 55 K and 30 K were the major synaptic vesicle calmodulin binding proteins. In addition there were two minor calmodulin binding singlet polypeptides with MrS 62 K and 40 K. Calmodulin stimulated endogenous synaptic vesicle protein kinase, Ca2+, Mg2+-ATPase and Ca2+ uptake activities. Phosphorylation assays coupled with immunological studies using affinity-purified antibodies suggested that the synaptic vesicle Ca2+/calmodulin-dependent protein kinase migrated in the 30 K Mr region.

Animals↗
PubMed1983

Characterization of brain synaptic vesicle phospholipase A2 activity and its modulation by calmodulin, prostaglandin E2, prostaglandin F2 alpha, cyclic AMP, and ATP.

Brain synaptic vesicle phospholipase A2 (PLA2) activity was characterized. It is Ca2+-dependent and has a pH optimum of 9.0. The enzyme has a Km of 60 microM and a Vmax of 2.0 nmol/mg/h. Calmodulin, prostaglandin F2 alpha, and cAMP, and ATP all increased the Vmax of the enzyme. Prostaglandin E2 inhibited the Vmax in the presence or absence of calmodulin. Light-scattering techniques in conjunction with phase-contrast and electron microscopy demonstrated that an increase in Vmax of PLA2 was correlated with synaptic vesicle aggregation, lysis, and possible fusion. In vitro synaptic vesicle-vesicle association that was stimulated by conditions that increased PLA2 activity could be diminished when synaptic vesicles were preincubated with PLA2 inhibitors. It is suggested that endogenous synaptic vesicle PLA2 activity may be an important mechanism underlying Ca2+-mediated neurotransmitter release.

Adenosine Triphosphate↗
PubMed1983

Phosphorylation of brain synaptic and coated vesicle proteins by endogenous Ca2+/calmodulin- and cAMP-dependent protein kinases.

Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5-15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity-purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin-dependent protein kinase. Affinity-purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin-dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP-dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000, 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.

Animals↗
PubMed1982

Comparison of calmodulin binding to brain synaptic and coated vesicles.

Several characteristics of calmodulin association with brain synaptic and coated vesicles wer analyzed and compared. Radioimmunoassay revealed that both classes of vesicles contain approx. 1 microgram of calmodulin per mg of vesicle protein. Discontinuous sucrose gradients revealed that coated and synaptic vesicles preparations were homogeneous and had different sedimentation properties. Binding of 125I-labeled calmodulin to sympatic and coated vesicles was Ca2+ dependent and displaced by unlabeled calmodulin but not by troponin-C. Scatchard analysis revealed the presence of two binding sites. In both vesicle types there was one high-affinity, low-binding-capacity site (Kd = 1-39 nM and Bmax = 4-16 pmol/mg) and one low-affinity, high-binding-capacity site (Kd = 102-177 nM and Bmax = 151-202 pmol/mg). (Ca2+ +Mg2+)-ATPase activity was stimulated in both synaptic and coated vesicles by calmodulin. Thus synaptic and coated vesicles may possess similar calmodulin binding sites.

Animals↗
PubMed1982

Interaction of brain synaptic vesicles induced by endogenous Ca2+ -dependent phospholipase A2.

Endogenous phospholipase A2 activity of brain synaptic vesicles was Ca2+ -dependent and was increased by prostaglandin F2 alpha, calmodulin, adenosine 3', 5' -monophosphate, and adenosine triphosphate, whereas the activity was inhibited by prostaglandin E2 in the absence or presence of calmodulin. Light-scattering measurements demonstrated that stimulation of the enzyme's activity correlated with the induction of vesicle-vesicle aggregation. The effects of these compounds on endogenous synaptic vesicle phospholipase A2 activity may imply a common end point of their purported neuromodulatory actions, and indicate that synaptic vesicle phospholipase A2 may play a central role in presynaptic neurotransmission.

Adenosine Triphosphate↗
PubMed1982

Brain clathrin and clathrin-associated proteins.

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.

Animals↗
PubMed1982

Brain clathrin: studies of its ultrastructural assemblies.

Clathrin purified to a high degree of homogeneity showed the presence of accompanying polypeptides of lower molecular weights and assembled into baskets or cages when the pH of its solution was adjusted from pH 7.5 to 6.5. Brief chymotrypsin treatment of this clathrin preparation at pH 6.5 cleaved clathrin-associated proteins rendering clathrin unable to reform baskets. Hydrolysis of clathrin-associated proteins did not affect clathrin's capacity to polymerize into open lattices or to bind actin and alpha-actinin from solution. When open lattices formed, the turbidity of the solution rose to levels that were higher than those produced when cages or baskets were formed by native clathrin. In both native or enzyme-treated clathrin, turbidity increase was inhibited by salts. The addition of certain cytoskeletal-disrupting agents, such as chlorpromazine and imipramine, increased clathrin's turbidity but did not result in formation of the baskets. Colchicine and cytochalasin B did not affect turbidity or the assembly of cages. The addition of increasing amounts of vinblastine resulted in the precipitation of larger amounts of clathrin which, after solubilization, retained its ability to polymerize into baskets. It seems that clathrin exists as a complex with other protein molecules, clathrin-associated proteins, that modulate the extent of polymerization and assembly the clathrin into characteristic structures.

Actinin↗
PubMed1982

Isolation and preliminary characterization of clathrin-associated proteins.

Clathrin-associated proteins were separated from clathrin under various clathrin-denaturing conditions, i.e. heating, freezing and isoelectric precipitation. The proteins retained biological activity; they were purified further by affinity chromatography on calmodulin-conjugated CNBr-Sepharose 4B and used for antibody purification. The affinity-purified anti-(clathrin-associated proteins) antibodies gave a fluorescent dotted pattern in cultured fibroblasts consistent with the known distribution of clathrin. Chemical cross-linking of pure clathrin-associated proteins indicated that these polypeptides exist as monomers in solution, each possessing Ca2+-dependent affinity for calmodulin to which they bind in a 1:1 molar ratio. Chymotryptic treatment of coated vesicles selectively cleaved the clathrin-associated proteins into a 15 000-18 000-Mr doublet polypeptide. These subfragments retained their Ca2+-dependent affinity for calmodulin. Our results support a regulatory role for clathrin-associated proteins in clathrin assemblies.

Calcium↗
PubMed1982

Calmodulin affinity for brain coated vesicle proteins.

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.

Animals↗
PubMed1980

Brain clathrin. Viscometric and turbidimetric properties of its ultrastructural assemblies.

Clathrin was isolated in highly purified form from bovine brain preparations rich in coated vesicles and by some improvements of our previous procedures. At pH 7.5, clathrin's solution was viscous, but clear. At pH 6.5, clathrin's solution was less viscous, but turbid. By electron microscopy, clathrin's turbidity at pH 6.5 correlated with the presence of numerous basket-like lattices or cages; the higher viscosity observed at pH 7.5 correlated with a mixture of various polymeric forms of clathrin having linearly assembled filaments or filamentous bundles of cross-linked clathrin molecules. In vivo, clathrin's capacity for assembling or disassembling itself into baskets or cage-like structures is compatible with a mechanism that retrieves areas of the plasma membrane containing protein molecules, smaller stimulatory or inhibitory compounds bound on the external cell membrane surface.

Animals↗
PubMed1980

Brain clathrin: immunofluorescent patterns in cultured cells and tissues.

High-purity bovine brain clathrin elicited antibodies, which were separated by affinity chromatography on clathrin conjugated with CNBr-activated Sepharose 4B. Indirect immunofluorescent labeling on cultured rat fibroblasts showed the characteristic dotted fluorescence throughout the entire cytoplasm and concentrated patches of fluorescence in areas where the Golgi apparatus and GERL (Golgi apparatus-endoplasmic reticulum-lysosome complex) exist next to the nucleus. Cerebellar tissue sections exhibited profuse immunofluorescence in the granular layer; dissociated cultures of neonatal rat cerebellar cells showed intense fluorescence in the cytoplasm and processes of granule cell neurons. The data suggest that clathrin is an abundant protein distinctly localized in the cytoplasm of cells and clearly correlated in its distribution and arrangement with actin stress fibers.

Animals↗
PubMed1979

Mechanochemical properties of brain clathrin: interactions with actin and alpha-actinin and polymerization into basketlike structures or filaments.

Two molar urea (pH 7.5) and column chromatography on Sepharose 4B were used to separate clathrin (coat protein) from the membrane of coated vesicles from bovine brain. Lytron (polystyrene) particles were used for study of the interaction of clathrin with contractile proteins. Muscle G-actin, F-actin, and alpha-actinin were bound by clathrin-coated Lytron particles, while no interaction was found when muscle tropomyosin and serum albumin were tested. Clathrin molecules dispersed in a solution of 20 mM Tris-HCl (pH 7.5) were found to be elongated. When the pH was adjusted from 7.5 to 6.5, clathrin molecules associated into basketlike or cage structures similar in size and shape to those observed in enriched preparations of coated vesicles. Below pH 6.0, cages or baskets became amorphous aggregates. Raising the pH from 6.5 to 8.0, addition of 5-10 mM ATP or EDTA, or addition of 200 mM KCl resulted in the dissassembly of baskets and the formation of filamentous arrays of various widths. Because of clathrin's biochemical and biophysical properties, its interaction with contractile proteins, and its presence in the membrane of vesicles of various cell types, we classified clathrin in the group of mechanochemical proteins.

Actinin↗