Biomedical subjects

N Moskowitz

Publications and source records attributed to N Moskowitz.

32 records · Page 2Linked to original sources

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↗

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↗

PubMed1979