Novel regulatory role of phosphorylated clathrin light chain beta in bovine brain coated vesicles.

The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs.

Clathrin-coated vesicle subtypes in mammalian brain tissue: detection of polypeptide heterogeneity by immunoprecipitation with monoclonal antibodies.

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton.

Brain coated vesicle destabilization and phosphorylation of coat proteins.

Two basic polypeptides, bee venom melittin and poly-L-lysine, induced concentration-dependent destabilization of bovine brain coated vesicles. Ultrastructurally the changes observed were aggregation of clathrin coats and segregation of the vesicle membrane, concomitant with the appearance of elongated cisternae of various sizes. Changes in coated vesicle morphology induced by melittin and poly-L-lysine were concurrent with stimulation of phosphate incorporation in proteins of the coat lattice: Mr 33,000 and 100,000.

Phosphorylation characteristics of brain clathrin-coated vesicle endogenous proteins.

Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g.

Mapping two functional domains of clathrin light chains with monoclonal antibodies.

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.