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S Puszkin

Publications and source records attributed to S Puszkin.

At least 37 records · Page 2Linked 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

Brain protein kinase in synaptic vesicles is inhibited by a factor in sera from systemic lupus erythematosus patients with central nervous system manifestations.

Sera from systemic lupus erythematosus patients with clinical central nervous system manifestations had a high mean percent of brain synaptic vesicle protein kinase inhibition (62 +/- 9.3, P less than 0.001). This conclusion was derived from screening sera of a total of 287 patients with heterogeneous diseases and from 12 healthy controls. Low levels of synaptic vesicle protein kinase inhibition also were detected in the sera of patients with certain autoimmune, inflammatory, neurological and/or psychiatric symptoms. Because 87.5% of the patients whose sera showed strong synaptic vesicle protein kinase inhibition (over 50%) had neuropsychiatric manifestations, we postulate this relationship may be due to the presence of an inhibitor factor, of which the etiology and molecular characteristics were investigated.

Adult↗
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↗
PubMed1986

Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I.

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.

Animals↗
PubMed1986

Calmodulin immunoelectron microscopy: redistribution during ram spermatogenesis and epididymal maturation. II.

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.

Animals↗
PubMed1985

Mathematical modelling of the formation of coated vesicles. A theoretical geometrical approach.

The mechanism by which flat hexagonal lattices of clathrin trimers transform into pentagonal/hexagonal spheres remains a mystery. In light of the geometrical nature of this process we have pursued a mathematical approach to the question. Through the geometrical analysis of flat hexagonal lattices we have discovered three possible forms of transformation to introduce curvature into the centre of the lattice: hub-centre transformation; hub-edge transformation; fringe transformation. Hub-edge and fringe transformations are used first to close the lattice while introducing localized curvature at the edges of the lattice. Hub-centre transformation is used after closure to relax the severely localized curvature generated during closure. This scheme not only maximizes the size of the coated vesicle generated, but also minimizes the number of transformations, thus minimizing the energy expended.

Clathrin↗
PubMed1985

A unified theory of presynaptic chemical neurotransmission.

The mechanism of neurotransmission and its modulation involves the direct role of calcium on membranes, and calcium's ability to activate synergistically and simultaneously a host of interdependent enzymatic cascades in synaptic and coated vesicles and the presynaptic plasma membrane. Enzymatic products formed can either amplify or depress synaptic vesicle exocytosis and synaptic vesicle regeneration via the coated pit/vesicle system. Rate amplification produced by a series of parallel, multistepped, interconnected enzymatic cascades as well as the optimal geometric spatial orientation of synaptic vesicles induced by presynaptic structures is hypothesized to explain how neurotransmitter is released within 200 musec upon calcium entry into the axon terminal.

Calcium↗
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↗
PubMed1985

Doxorubicin and covalently crosslinked doxorubicin derivatives binding to purified cardiac thin-filament proteins in vitro.

The binding of cardiac actin and tropomyosin to a cardiotoxic antineoplastic agent, doxorubicin, and its covalently crosslinked derivatives was investigated. The primary amino group of the daunosamine moiety of doxorubicin was blocked with fluorescein isothiocyanate. This doxorubicin derivative did not bind to Sepharose which was conjugated with cardiac actin. A doxorubicin dimer was made by covalently crosslinking one doxorubicin molecule to another identical doxorubicin molecule through the free amino group of each daunosamine moiety. This derivative demonstrated mobility different from parent doxorubicin on thin-layer chromatography, different elution pattern by column chromatography, and did not show binding affinity for actin. Exploring other purified thin-filament proteins, it was found that doxorubicin did bind to tropomyosin when gel filtration was performed on the protein drug mixture. The ability of tropomyosin to form paracrystal in vitro was not disturbed by a variety of concentrations of doxorubicin. These data support the concept that the doxorubicin solitary free amino group is the site which is responsible for this ligand to bind to actin and may relate to its cardiotoxic effects.

Actins↗
PubMed1985

Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.

A protein kinase activity was observed in coated vesicles, prepared from bovine brain, that had clathrin-associated protein 2 (CAP2, also known as clathrin light chain 2) as its principal substrate. Coated vesicles were purified by sucrose density gradient centrifugation followed by Sephacryl S-1000 column chromatography, and all buffers utilized in these procedures contained a mixture of proteolysis inhibitors to maintain CAP2 kinase activity. Incubation of vesicles with [gamma-32P]ATP in the presence of 7 microM polylysine resulted in an overall increase in the incorporation of phosphate. NaDodSO4/PAGE revealed that the principal recipient of this additional phosphate was CAP2 (Mr 33,000), the faster-migrating component of the clathrin coat-associated proteins, whereas CAP1 (Mr 36,000) was not phosphorylated. A number of other proteins, in the Mr 140,000 and 100,000 regions, were phosphorylated to a lesser extent. Polyarginine and polyethylenimine also supported CAP2 phosphorylation, but arginine and lysine were ineffective. The phosphorylated protein was identified as CAP2 because addition of exogenous CAPs resulted in increased incorporation of label into Mr 33,000 polypeptides and because heat treatment of labeled vesicles followed by ultracentrifugation resulted in recovery of labeled Mr 33,000 protein in the supernatant. Phosphorylation of CAP2 may play a regulatory role in clathrin coat/coated vesicle functions.

Animals↗
PubMed1984

Clathrin lattice reorganization: theoretical considerations.

We wish to postulate a mechanism by which flat hexagonal lattices of clathrin trimers transform into coated pits. Using an established model for packing trimers into lattices, we explored the assembly process by single addition of trimers to form polygons. Subject to favorable conditions, removal of a single trimer from a hexagon could lead to the formation of a pentagon. Elimination of trimers from polygonal sheets can occur either at the center of the network or at the edges. Removal of a trimer from the center of these adjacent polygons, "hub transformation," is possible in very few instances, whereas removal from the edges of a polygonal sheet, "fringe transformation," is possible in a host of cases. These hypothetical constructs can be used effectively to explain intermediate structures actually observed in flat hexagonal lattices. The geometry of a purely hexagonal lattice seems to dictate that the first step in transformation must be a "fringe transformation," which then will allow subsequent "hub transformation" to take place leading to the introduction of pentagons into the center of the lattice and ultimately to the curvature of the clathrin lattice.

Biopolymers↗
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↗
PubMed1984

Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture.

Bovine chromaffin cells maintained in culture for eight days were loaded with [3H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K+. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus. The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K+ produced 91 +/- 2% (n = 5) depletion in the [3H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1983

Clathrin-associated proteins contain bound nucleotide.

Clathrin-associated proteins purified from bovine brain exhibited an ultraviolet spectrum with absorbance maximum at 256 nm and were found to contain tightly bound nucleotide. This nucleotide was identified as AMP and/or ADP by thin-layer and high-performance liquid chromatographic analyses. The phosphorylation state of the bound nucleotide varied with storage conditions, suggesting that exchange with ATP might occur while a molar ratio of two nucleotides per protein molecule is maintained. This nucleotide binding site may play a role in the functions of clathrin-associated proteins.

Adenine Nucleotides↗
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

Immunocytochemical characterization of clathrin-associated proteins (CAPs). I. Neuronal distribution of CAPs, a component of clathrin-coated vesicles.

Clathrin-associated proteins (CAPs) elicited antibodies in rabbits that were affinity-purified using CAPs-conjugated CNBr-Sepharose 4B. Anti-CAPs IgG formed immunoprecipitates with CAPs and with the clathrin-CAPs complex. Indirect immunoperoxidase-labeling in sections of rat brain cortex using anti-CAPs and anti-clathrin IgG yielded similar staining patterns. Coated perinuclear cisternae and coated vesicles became stained and easily distinguished. Intense staining also was found in synaptic boutons with label between most synaptic vesicles and as a thick crust surrounding coated vesicles. The data demonstrate that clathrin and CAPs polypeptides are in identical subcellular locations.

Animals↗