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S Puszkin

Publications and source records attributed to S Puszkin.

At least 55 records · Page 3Linked to original sources
PubMed1983

Immunocytochemical characterization of clathrin-associated proteins (CAPs). II. Immunolocalization of CAPs using selectively-absorbed IgG solutions.

Utilizing antibodies elicited by clathrin-associated proteins (CAPs) absorbed with three different antigenic states of CAPs, i.e., bound to clathrin (clathrin-CAPs complex), free in solution (CAPs) or partially cleaved by chymotrypsin (CAPs-subfragments), indicated that when CAPs are bound to clathrin an antigenic site (or sites) remain(s) unexposed and CAPs-subfragments lose antigenic sites as a result of limited proteolysis. IgG remaining in solution after absorption with CAPs-subfragments were directed against the chymotrypsin-sensitive, or accessible portions of CAPs, whereas IgG remaining after absorption with clathrin-CAPs complex were directed against the unexposed antigenic site(s) characteristic of the clathrin-CAPs complex. Immunocytochemical characterization of these selectively-absorbed IgG solutions suggests that CAPs detected during immunolocalization exist as a complex with clathrin.

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↗
PubMed1983

Compartmentalization of adriamycin and daunomycin in cultured chick cardiac myocytes. Effects on synthesis of contractile and cytoplasmic proteins.

The affinity of two anthracycline antineoplastics [( 14C]adriamycin and [14C]daunomycin) for cultured embryonic chick heart cells was determined by measuring their uptake, compartmentalization into subcellular fractions, and effects on the synthesis of cytoplasmic and cytoskeletal proteins. Both drugs, at micromolar concentrations, were readily uptaken by myocytes and found to be concentrated in a light-buoyant-density fraction containing no lysosomes. Nuclear 14C drug content accounted for 20-25% of the drug incorporated. Binding of adriamycin was saturable within 90 minutes of drug exposure, and the uptake of [14C]adriamycin was inhibited 50% by verapamil and adenosine triphosphate. Uptake of [14C]daunomycin was not influenced by these compounds. Cytosolic and contractile protein synthesis measured by [35S]methionine incorporation into proteins was blocked 70% overall in each fraction after 6 hours of incubation with 2 microM adriamycin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography and quantitative densitometry, revealed that actin synthesis was the least affected of the major proteins. Cardiac myocytes incubated for short periods of time with 2 microM adriamycin revealed subtle cytoplasmic changes in their organelles with the appearance of clear zones of cytoplasm containing short unorganized microfilaments. The deleterious effects of anthracyclines in heart cells are manifested early by rapid drug incorporation into myocytes and inhibition of cytoplasmic protein synthesis.

Animals↗
PubMed1983

Brain clathrin: immunofluorescent localization in rat retina.

By indirect immunofluorescence, the major protein of coated vesicles and coated pits was localized in rat retina. Specific fluorescence was circumscribed to regions containing synaptic endings in high density such as the cell internal plexiform layer (IPL) and external plexiform layer (EPL). Fine dots or patches of fluorescence were seen in the EPL. Weaker fluorescence was observed in the narrow cytoplasmic strips surrounding the large nucleus of the ganglion cell, internal nuclear and external nuclear cell layers. There was no fluorescent staining in the retinal pigment epithelium, a layer where active endocytosis of the outer segment of the rods and cones take place. The high concentration of clathrin found in the IPL and EPL is indicative of active recycling of synaptic membrane concomitant to neurotransmitter release and receptor molecule turnover.

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

Immune localization of calmodulin in the ciliated cells of hamster tracheal epithelium.

Melachronous beating of cilia of epithelial surfaces of most respiratory airways moves the overlying mucous layer in a caudal direction. The molecular mechanisms controlling ciliary beat remain largely unknown. Calcium, an element in its cationic form, is ubiquitous in biological functions and its concentration is critical for ciliary beating. Calmodulin, a calcium-binding protein which regulates the activity of many enzymes and cellular processes, may regulate ciliary beating by controlling enzymes responsible for mechanochemical movement between adjacent peripheral microtubule doublets composing the ciliary axoneme. As a first step in describing a calmodulin-related controlling mechanism for ciliary beating, calmodulin was localized in the ciliated cells lining the respiratory tracts of hamsters by electron microscopy, using an indirect immunoperoxidase technique with anticalmodulin antibodies as the molecular probe. Thin-sections revealed calmodulin located on microtubules and dynein arms of the ciliary shaft, basal body, apical cytoskeletal microtubules, and plasma membranes in specimens fixed with 1 mM Ca+2. Specimens fixed with less Ca+2 (1 microM), Mn+2, Mg+2, and EGTA showed a diffuse pattern of calmodulin with loci of greatest densities on basal body microtubule triplets. Demembranated specimens showed a less specific localization on axonemal microtubules but only on cells fixed with Ca+2. Calmodulin, by binding calcium, may function in ciliary beating in the respiratory tract of mammals either directly or indirectly through its effects on the energy-producing enzymes and by control of Ca+2 flux through plasma membranes.

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↗
PubMed1982

Interaction of adriamycin in vitro with cardiac myofibrillar proteins.

Binding of the anthracycline antibiotic doxorubicin [Adriamycin (ADR)] to selected cardiac muscle contractile proteins was determined in purified canine heart actin and alpha-actinin. Adriamycin binding to these proteins in solution was measured by equilibrium dialysis and gel filtration with [14C]-labeled ADR. Adriamycin did not bind when its primary amino group was blocked. Adriamycin binding did not affect actin polymerization as detected by viscometry. Viewed under the electron microscope, however, ADR promoted formation of distinct actin filaments in the presence of microM amounts of ATP without K+ and MG++. Adriamycin-induced actin microfilaments were thicker (120 A) than those of F-actin controls (70 A) and stimulated myosin-ATPase to higher levels than the F-actin controls. It appears that ADR has a direct effect on the biophysical and biochemical properties of heart myofibril proteins in vitro.

Actinin↗
PubMed1982

Brain clathrin complex: II. Immunofluorescent correlation and biochemical affinity for actin.

The interaction of clathrin with cytoskeletal proteins was studied cytochemically by immunofluorescent staining and biochemically by the binding of actin to clathrin on the surfaces of polystyrene particles. Using a cytoskeletal-disrupting agent, the linear arrangement of clathrin lattices along actin fibers was altered. As a result of cell retraction, the fluorescent dots of clathrin redistributed, conforming to the new cellular shape. Cytoplasmic areas, largely devoid of fluorescent dots, were observed at the cell's periphery. In vitro, the native clathrin complex (clathrin plus clathrin-associated proteins (CAPs)) bound up to 1 mol of actin, but when the clathrin polypeptide was separated from accompanying proteins it bound up to 2 mol of actin from solution. It appears that clathrin's molecular lattices have an affinity for arrays of actin microfilaments, following them closely, and that clathrin lattices display lateral mobility during cytoplasmic reorganization.

Actins↗
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↗